KR20080049014A - Human anti-b7rp1 neutralizing antibodies - Google Patents

Abstract

This invention provides antibodies that interact with or bind to human B7 related protein-1 (B7RP1) and antibodies that bind to and neutralize the function of B7RP1 thereby. The invention also provides pharmaceutical compositions of said antibodies and methods for neutralizing B7RP1 function, and particularly for treating immune disorders (e.g., inappropriate immune response) by administering a pharmaceutically effective amount of anti-B7RPl antibodies. Methods of detecting the amount of B7RP1 in a sample using anti-B7RPl antibodies are also provided.

Description

Human anti-VII7P1 neutralizing antibody {HUMAN ANTI-B7RP1 NEUTRALIZING ANTIBODIES}

This application was filed on July 18, 2005. Provisional Patent Application Serial No. 60 / 700,265 is priority, the disclosure of which is hereby incorporated by reference in its entirety.

The present invention relates to human monoclonal antibodies that bind to B7 related protein-1 (B7RP1). Also described are compositions and methods for treating diseases and disorders associated with immunosuppression and immune activation.

T-cells initiate an immune response, mediate antigen-specific effector function, and regulate the activity of other white blood cells by cytokine secretion. In order to generate an appropriate T-lymphocyte (T-cell) immune response, two signals must be provided to T-cells by antigen presenting cells (APC). The antigen must be presented to the T-cell receptor (TCR) via the major histocompatibility gene complex (MHC) when determining specificity. T cells can only recognize antigens presented on APC. In addition to antigen receptors, proper T-cell activation also requires the interaction of other cell surface molecules in both T-cells and APCs. These molecules, referred to as costimulatory molecules, consist of a ligand present on the inducer cell and a receptor on the response cell. The antigen independent costimulatory signal must be delivered by the association of members of the B7 family on APC and its receptors on T-cells. Productive immune responses induce proliferation, differentiation, clonal expansion and effector function. Without a second costimulatory signal, T-cells are placed in a state of long-lasting antigen-specific non-response, called immunogenicity. Phase II clinical trials, psoriasis (Abrams et al. 2000, J Exp Med . 192: 681-94; Abrams et al . 1999, J. Clin . Invest . 103: 1243-52) and rheumatoid arthritis (Kremer et al., 2003, New England Journal of Medicine 349: 1907-15), it is demonstrated that blocking one co-stimulatory pathway is effective, suggesting that this general strategy is a good target for immunomodulatory therapy.

A special costimulatory B7 molecule, B7 related protein-1 (B7RP1), is a type 1 transmembrane protein with a signal sequence and an extracellular domain at its amino terminus, wherein the extracellular domain is composed of two Ig loops, a transmembrane domain and Carboxy terminal intracellular domains (PCT Application Publication WO 00/46240). B7RP1 is inducible to ICOS expressed on the cell surface of T-cells. Display an inducible costimulator] (Yoshinga et al . 2000, Int . Immun. 12: 1439-1447). ICOS plays an important role in the production of type 1 and type 2 cytokines by activated T-cells (Coyle et al. 2000, Immunity 13: 95-105).

B7RP1 is the only ligand that is constantly expressed on APC (1999, Nature . 402: 827-32 by Yoshinaga et al . ) And ICOS is expressed only on activated T-cells (McAdam et al. 2000, Journal of Immunology 165: 5035-40). B7RP1-dependent signaling is required for the activation of effector (ie fully activated) T-cells as well as for their maturation from their naïve precursors (Dong et al., 2003, Journal of Autoimmunity . 21: 255-60; Coyle 2000, Immunity . 13: 95-105). As a result, B7RP1 / ICOS interactions are required for proper T-cell-dependent calling immune responses (Dong et al., 2003, Journal of Autoimmunity . 21: 255-60).

Recent attempts to disrupt co-stimulatory T-cell pathways have focused on co-stimulatory polypeptides that block only T-cell activation, but not activation and maturation. As a result, these therapies provide general inhibition of T-cell function. In contrast, blocking of the B7RP1 / ICOS interaction provides more specific inhibition of T-cell function affected only by mature effector T-cells. Accordingly, blocking of B7RP1 / ICOS interactions in clinical settings is even more desirable as it provides a more limited side effect profile compared to costimulatory therapy that blocks only natural T-cell activation.

Summary of the Invention

The present invention provides monoclonal antibodies that bind to B7 related protein-1 (B7RP1). In one embodiment, the monoclonal antibody is a human monoclonal antibody that is particularly useful for neutralizing the biological activity of B7RP1 and specifically or completely inhibiting the immune costimulatory activity of B7RP1. The invention also provides cells, in particular hybridoma cells, which produce the monoclonal antibodies of the invention. In a particular aspect, the antibodies of the invention specifically bind to the H or D region of B7RP1 as described herein.

The invention further provides a fusion protein comprising a sequence of an antibody Fc region and at least one sequence identified by SEQ ID NO: 1 to SEQ ID NO: 40. Such molecules can be prepared, for example, using the methods described in international patent application, publication WO 00/24782, which is incorporated herein by reference. Such molecules can be, for example, mammalian cells (eg, Chinese hamster ovary cells) or bacterial cells [eg, Esherikia Colai ( E. coli ) cells.

In certain aspects, the invention relates to antibodies comprising heavy and light chains, wherein the heavy chains comprise a heavy chain constant region or any allelic variant thereof selected from IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE heavy chain constant regions. (As discussed in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, which is incorporated herein by reference). An antibody is provided comprising the amino acid sequence set forth in any of SEQ ID NOs: 7 to SEQ ID NO: 14, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment. Antibodies of the invention comprise the amino acid of any heavy chain constant region of IgG2 shown in SEQ ID NO: 41 or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment, or an IgG1 heavy chain shown in SEQ ID NO: 42 Amino acid sequences of the constant region or antigen-binding fragments thereof or immunologically functional immunoglobulin fragments. In certain embodiments, the antibody is a monoclonal antibody, a human antibody, or preferably a human monoclonal antibody.

In certain aspects, the present invention provides antibodies comprising heavy and light chains, wherein the light chain is a constant region having an amino acid sequence set forth in SEQ ID NO: 43 or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment Wherein the light chain variable region comprises the amino acid sequence set forth in any of SEQ ID NOs: 1 to SEQ ID NO: 6, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment. In certain embodiments, the antibody is a monoclonal antibody, a human antibody, or preferably a human monoclonal antibody.

In certain aspects, antibodies of the invention include heavy and light chains, wherein the variable region of the heavy chain is an amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 8, or an antigen binding fragment thereof or immunologically functional Immunoglobulin fragments. In other aspects, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment.

In other aspects, antibodies of the invention include heavy and light chains, wherein the variable region of the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 9, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment . In other aspects, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 2, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment.

In a further aspect, the heavy chain comprises at least one complementarity determining region (CDR) having the amino acid sequence set forth in any of SEQ ID NOs: 27 to SEQ ID NO: 40, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment. It includes. In a further aspect, the light chain comprises at least one CDR having the amino acid sequence set forth in any of SEQ ID NOs: 15 to SEQ ID NO: 26, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment.

The invention also provides an antigen that specifically binds to B7RP1, wherein the heavy chain is an amino acid sequence represented by SEQ ID NO: 7 or SEQ ID NO: 8, or an antigen binding fragment or immunologically functional immunoglobulin fragment thereof Wherein the light chain comprises a variable region comprising an amino acid sequence set forth in SEQ ID NO: 1, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment.

In addition, the present invention provides antibodies that specifically bind to B7RP1, wherein the heavy chain comprises a variable comprising an amino acid sequence set forth in SEQ ID NO: 9, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment Wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO: 2, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment.

In certain aspects, the invention also provides antibodies comprising heavy and light chains, wherein the heavy chain comprises a heavy chain variable region, wherein the heavy chain variable region is set forth in any of SEQ ID NOs: 7 to SEQ ID NO: 14 Identity to an amino acid sequence as such is at least about 75% or more, at least about 80% or more, at least about 85% or more, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or more, or at least about 99% or more, wherein the light chain comprises a light chain variable region, wherein the light chain variable region is as set forth in any of SEQ ID NOs: 1 to SEQ ID NO: 6 At least about 80%, at least about 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more, or at least about Contains at least 99% sequence, , Where, the antibody binds specifically to B7RP1.

The invention also relates to an antibody that specifically binds to B7RP1, wherein the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 44 or SEQ ID NO: 46, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment And an antibody comprising an amino acid sequence as set forth in SEQ ID NO: 45, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment.

The invention also provides an antibody that specifically binds to B7RP1, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 47, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment, wherein the light chain is Amino acid sequence set forth in SEQ ID NO: 48, or an antigen binding fragment or immunologically functional immunoglobulin fragment thereof.

In certain aspects, the invention provides antibodies comprising heavy and light chains, wherein the heavy chain comprises a heavy chain variable region, wherein the heavy chain variable region is an amino acid sequence set forth in any of SEQ ID NOs: 27 to SEQ ID NO: 40. And at least about 75% or more, at least about 80% or more, at least about 85% or more, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more, or At least one CDR sequence having at least about 99% or more identity, the light chain comprising a light chain variable region, wherein the light chain variable region is at least about 80 with the amino acid sequence set forth in SEQ ID NOs: 15 to SEQ ID NO: 26 An amino acid sequence having at least%, at least about 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% identity Contains at least one CDR with And, wherein the antibody binds specifically to B7RP1.

The invention also provides single chain antibodies, single chain Fv antibodies, F (ab) antibodies, F (ab) antibodies and (Fab ') ₂ antibodies.

In a particular aspect, the present invention provides a light chain comprising the amino acid sequence set forth in SEQ ID NO: 15 to SEQ ID NO: 26, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment.

In addition, the present invention provides a heavy chain comprising the amino acid sequence set forth in any of SEQ ID NOs: 27 to SEQ ID NO: 40, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment.

The invention also relates to an isolated human antibody that specifically binds to B7RP1, comprising: (a) a human heavy chain framework region, a human heavy chain CDR1 region, a human heavy chain CDR2 region, and a human heavy chain CDR3 region; And (b) human light chain framework regions, human light chain CDR1 regions, human light chain CDR2 regions, and human light chain CDR3 regions. In certain aspects, the human heavy chain CDR1 region can be the heavy chain CDR1 region as shown in any SEQ ID NO: 27, 30 or 35, and the human light chain CDR1 region as the light chain CDR1 region as shown in any SEQ ID NO: 15, 18 or 24. Can be. In other aspects, the human heavy chain CDR2 region can be the heavy chain CDR2 region shown in any SEQ ID NO: 28, 31, 33, 36, or 39, and the human light chain CDR2 region can be in any SEQ ID NO: 16, 19, or 21. Light chain CDR2 shown. In another aspect, the human heavy chain CDR3 region is a heavy chain CDR3 region as shown in any SEQ ID NO: 29, 32, 34, 37, 38, or 40, and the human light chain CDR3 region is any SEQ ID NO: 17, 20, 22 , Light chain CDR3 region as shown in 23, 25 or 26.

Antibodies of the invention are characterized by their ability to specifically bind B7RP1. Moreover, the antibodies of the present invention have the ability to antagonize at least one of the in vitro (or ex vivo) and / or in vivo activity associated with the B7RP1 polypeptide. The invention wherein the separation with high affinity binding to B7RP1 polypeptide to provide a human B7RP1 human antibody, wherein the antibody is about 10 ^-6 M, 10 ^-7, and is coupled to a human B7RP1 polypeptide, determined using the KinExA M, 10 ^-8 M, 10 ^-9 M, 10 ^-10 M, 10 ^-11 M, 10 ^-12 M dissociated from the human B7RP1 polypeptide with a dissociation constant (K _D ), or about 10 ^-6 M, 10 ^-7 M, inhibit ^{10 -8 M, 10 -9 M,} 10 -10 M, 10 -11 M, 10 -12 M the degree of B7RP1 induced survival in an in vitro neutralization test as EC _50.

The invention also provides an isolated human antibody or antigen-binding fragment or immunologically functional immunoglobulin fragment that specifically binds to B7RP1, wherein the antibody or fragment is a heavy chain variable region comprising heavy chain CDR1, CDR2 and CDR3 A) heavy chain CDR1 has an amino acid sequence as shown in SEQ ID NO: 27, heavy chain CDR2 has an amino acid sequence as shown in SEQ ID NO: 28, and heavy chain CDR 3 has a SEQ ID NO: Has an amino acid sequence set forth at 29; b) heavy chain CDR1 has the amino acid sequence shown in SEQ ID NO: 30, heavy chain CDR2 has the amino acid sequence shown in SEQ ID NO: 31, and heavy chain CDR 3 has the amino acid sequence shown in SEQ ID NO: 32 ; c) heavy chain CDR1 has the amino acid sequence shown in SEQ ID NO: 27, heavy chain CDR2 has the amino acid sequence shown in SEQ ID NO: 33, and heavy chain CDR 3 has the amino acid sequence shown in SEQ ID NO: 34 ; d) heavy chain CDR1 has the amino acid sequence shown in SEQ ID NO: 35, heavy chain CDR2 has the amino acid sequence shown in SEQ ID NO: 36, and heavy chain CDR 3 has the amino acid sequence shown in SEQ ID NO: 37 ; e) heavy chain CDR1 has the amino acid sequence shown in SEQ ID NO: 27, heavy chain CDR2 has the amino acid sequence shown in SEQ ID NO: 33, heavy chain CDR 3 has the amino acid sequence shown in SEQ ID NO: 38 ; Or f) heavy chain CDR1 has the amino acid sequence shown in SEQ ID NO: 35, heavy chain CDR2 has the amino acid sequence shown in SEQ ID NO: 39, and heavy chain CDR 3 has the amino acid sequence shown in SEQ ID NO: 40 Have

The invention also provides human antibodies or isolated human antibodies or antigen binding fragments thereof or immunologically functional immunoglobulin fragments that specifically bind to B7RP1, wherein the antibody or fragment comprises light chain CDR1, CDR2 and CDR3. A light chain variable region, as follows: a) Light chain CDR1 has the amino acid sequence shown in SEQ ID NO: 15, light chain CDR2 has the amino acid sequence shown in SEQ ID NO: 16, and light chain CDR3 has the SEQ ID NO: has the amino acid sequence set forth in 17; b) light chain CDR1 has the amino acid sequence shown in SEQ ID NO: 18, light chain CDR2 has the amino acid sequence shown in SEQ ID NO: 19, and light chain CDR3 has the amino acid sequence shown in SEQ ID NO: 20; c) light chain CDR1 has the amino acid sequence shown in SEQ ID NO: 15, light chain CDR2 has the amino acid sequence shown in SEQ ID NO: 21, and light chain CDR3 has the amino acid sequence shown in SEQ ID NO: 22; d) light chain CDR1 has the amino acid sequence shown in SEQ ID NO: 18, light chain CDR2 has the amino acid sequence shown in SEQ ID NO: 19, and light chain CDR3 has the amino acid sequence shown in SEQ ID NO: 23; e) light chain CDR1 has the amino acid sequence shown in SEQ ID NO: 24, light chain CDR2 has the amino acid sequence shown in SEQ ID NO: 16, and light chain CDR3 has the amino acid sequence shown in SEQ ID NO: 25; Or f) light chain CDR1 has the amino acid sequence shown in SEQ ID NO: 24, light chain CDR2 has the amino acid sequence shown in SEQ ID NO: 16, and light chain CDR3 has the amino acid sequence shown in SEQ ID NO: 26. .

The invention also provides antibodies that compete with the antibodies described herein for B7RP1. In certain aspects, a competitive antibody of the invention competes for binding to human B7RP1 of an antibody comprising any of SEQ ID NOs: 1-40.

In addition, some of the present invention incorporate a polynucleotide sequence encoding an anti-human B7RP1 human antibody, a vector comprising a polynucleotide sequence encoding an anti-human B7RP1 human antibody, a polynucleotide encoding an anti-human B7RP1 human antibody Host cells, anti-human B7RP1 human antibodies transformed with the vector, and methods for making and using the same.

The present invention also provides a method for detecting B7RP1 in a biological sample, comprising contacting the sample with an antibody of the invention or antigen-binding fragment thereof. The anti-B7RP1 antibodies of the invention can be used in any known assay for B7RP1 detection and calibration, such as competitive binding assays, direct and indirect sandwich assays, immunoprecipitation assays and enzyme-linked immunosorbent assays (ELISA). (See, Sola, 1987, Monoclonal Antiboeis: A Manual of Techniques, pp. 147-158, CRC Press, Inc.). The antibody may bind to B7RP1 with an affinity suitable for the assay used.

In addition, the present invention comprises administering to an individual in need thereof an effective amount of a pharmaceutical composition containing at least one antibody or antigen-binding fragment thereof or immunologically functional immunoglobulin fragment of the present invention. A method of treating transpiration, disease associated with increased sensitivity to B7RP1 and / or disease associated with modulating T-cell response.

Embodiments of the invention will be apparent in the following detailed description and claims.

1A shows the 16H antibody variable region sequence (SEQ ID NO: 7) and the corresponding 16H variable region germline (16Hg) sequence (SEQ ID NO: 8).

1B depicts the results of the costimulatory assay using anti-CD3 and hB7RP1-Fc fusion proteins demonstrating that 16Hg retains its biological activity comparable to 16H.

결합 검정 결과를 보여준다.2 shows Biacore with 16H, 16Hg, and 5D antibodies

Show binding test results.

3 shows the results of a KinExA binding assay using 5D antibodies.

4 shows the KinExA binding assay results using 2H antibodies.

5 shows KinExA binding assay results using 2H germline (2Hg) antibodies.

FIG. 6 shows binding-competition assay results showing that 16H antibodies compete with binding of ICOS-Fc to B7RP-1, analyzed by flow cytometry.

7 shows an overview of B7RP-1 single nucleotide polymorphism (SNP) analysis.

8 shows an overview of the analysis of an anti-human B7RP-1 monoclonal antibody set in an ELISA competition assay. Values shown are IC ₅₀ for inhibition of binding of ICOS-Fc fusion protein.

9A shows fluorescence staining of B7RP1 extracellular domain (ECD) with labeled 16H, 5D and ICOS antibodies.

9B shows similar binding efficacy of 16H and 5D antibodies against B7RP1 SNP variants.

9C depicts the results of costimulatory assays using 16H or 5D antibodies and SNP variants.

10A shows plate costimulatory assay results using 1B7v2 monoclonal antibodies compared to a number of different anti-murine B7RP-1 monoclonal antibodies.

10B, 10C and 10D show the results of antigen challenge experiments analyzed for antigen-specific serum IgM (FIG. 10B), IgG2a (FIG. 10C) and IgG1 (FIG. 10D).

11 shows ELISA results demonstrating that serum IL-5 levels are inhibited with 1B7v2 antigen.

12A shows that 16H antibodies can bind to cynomolgus monkey B7RP1 (right panel) and human B7RP1 (left panel).

12B shows that 16H, 16Hg and 5D antibodies can inhibit cynomolgus monkey B7RP1 / ICOS-dependent T cell activation.

FIG. 13A depicts individual cynomolgus monkey and group mean titers on days 53 and 57 after secondary challenge with tetanus toxoid at day 42 in animals treated with 16H antibody.

13B depicts individual cynomolgus monkey and group mean titers on days 53 and 57 after secondary challenge with tetanus toxoid at day 42 in animals treated with 5D antibody.

Section headings used herein are for organizational purposes only and should not be construed as limiting the subject sanctions described. All references cited in this application are expressly incorporated herein by reference for any purpose.

Justice

Conventional techniques (eg, electrophoresis, lipofection) can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions or generally as described in the art or as described herein. The techniques and procedures can be generally performed as described in various general and more specific references cited and discussed herein, according to methods well known in the art. See, eg, 2001, MOLECULAR CLONING: A LABORATORY MANUAL, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Is incorporated herein by reference for any purpose. Unless otherwise defined, the nomenclature and laboratory procedures and techniques used in connection with the analytical chemistry, organic synthetic chemistry, and medicinal chemistry described herein are those well known and commonly used in the art. Similarly, conventional techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and patient treatment.

As used in accordance with the present disclosure, the following terms will be understood to have the following meanings, unless indicated otherwise. The phrases “biological properties”, “biological properties” and the term “activity” related to the antibodies of the invention are used interchangeably herein and include, but are not limited to, epitope affinity and specificity (eg, anti-human to human B7RP1). B7RP1 human antibody binding), the active antagonistic ability of the targeting polypeptide (eg, B7RP1 activity), the in vivo stability of the antibody and the immunogenic properties of the antibody. Other identifiable biological properties or characteristics of antibodies recognized in the art include, for example, mutual reactivity (eg, with non-human homologs of B7RP1, or generally with other proteins or tissues), and mammals. The ability to maintain high expression levels of proteins in cells. The above-mentioned properties or features include, but are not limited to, ELISA, competitive ELISA, surface plasmon resonance, in vitro and in vivo neutralization assays (eg, Example 2), and humans, primates or any other suitable source. It can be observed or measured using techniques known in the art, including immunostaining for tissue sections of. Particular activity and biological properties of the anti-human B7RP1 human antibody are described in more detail in the Examples below.

As used herein, the term “isolated polynucleotide” means a polynucleotide of genomic DNA, cDNA, RNA, or synthetic origin or any combination thereof, and according to its origin, an isolated polynucleotide is (1) an isolated poly If nucleotides are found in nature, they do not relate to all or part of the polynucleotide, (2) bind to a polynucleotide that is not bound in nature, or (3) occur as part of a larger sequence in nature.

As used herein, the term "polynucleotide" refers to a single stranded or double stranded nucleic acid polymer of at least 10 nucleotides in length. In certain embodiments, the nucleotides comprising the polynucleotides may be ribonucleotides or deoxyribonucleotides or modified forms of nucleotides of any type thereof. Such modifications include base modifications such as bromuridine, ribose modifications such as arabinoside and 2,3-dideoxyribose and internucleotide binding modifications such as phosphorothioate, phosphorodithioate, phosphorosenoate, Phosphorodiselenoates, phosphoroanilothioates, phosphoraniladates and phosphoramidates. The term "polynucleotide" includes specifically single and double stranded forms of DNA or RNA.

The term “oligonucleotide” herein includes naturally occurring oligonucleotides and modified nucleotides and / or non-naturally occurring oligonucleotide bonds that are linked to each other by naturally occurring. Oligonucleotides are polynucleotide subtypes that generally comprise members that are single stranded and have a length of about 200 nucleotides. In certain embodiments, oligonucleotides are 10 to 60 nucleotides in length. In certain embodiments, oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19 or 20 to 40 nucleotides in length. Oligonucleotides may be single- or double-stranded, eg, used for construction of gene mutations. Oligonucleotides of the invention can be sense or antisense oligonucleotides for protein coding sequences.

The term “naturally occurring nucleotides” includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotide" includes nucleotides having modified or substituted sugar groups, and the like. The term "oligonucleotide bond" includes oligonucleotide bonds, such as phosphorothioate, phosphorodithioate, phosphorosenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilate, phosphoro Amidate and the like. For reference, See, eg , LaPlanche et al . 1986, Nucl . Acids Res . , 14: 9081; Stec et al. 1984, J. Am . Chem . Soc . 106: 6077; Stein et al . 1988, Nucl . Acids Res . , 16: 3209; Zon et al. 1991, Anti - cancer Drug Design , 6: 539; 1991, OLIGONUCLEOTIDE AND ANALOGUES: A PRACTICAL APPROACH, pp. 87-108 (F. Eckstein, Ed.), Oxford University Press, Oxford England; US patent no. 5,151,510; Uhlmann and Peyman, 1990, Chemical Reviews , 90: 543, which disclosures are incorporated herein by reference for any purpose. Oligonucleotides may include detectable labels that allow detection of oligonucleotides or hybridization thereof.

As used herein, the term “isolated protein” means that the protein of interest is (1) free from at least some other proteins found in nature, or (2) essentially from other proteins of the same source, eg, from the same species. At least about 50% or more of the polynucleotides, lipids, carbohydrates, or other materials of interest, (3) are expressed by cells from different species, or (4) in nature are isolated, or (5) Protein is not associated with a portion of the protein that is involved in nature (by covalent or non-covalent interactions), or (6) with a polypeptide that is not involved in nature (by covalent or non-covalent interactions). Or (7) does not occur in nature. The isolated protein may be encoded by genomic DNA, cDNA, mRNA or other RNA, synthetic origin or any combination thereof. In one embodiment, an isolated protein is substantially advantageous from proteins or polypeptides or other contaminants found in its natural environment that interfere with its use (treatment, diagnosis, prevention, research or otherwise).

An “isolated” antibody is one that has been identified and isolated and / or recovered from members of its natural environment. Contaminated members of its natural environment are substances that would interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or nonproteinaceous substances. In certain embodiments, the antibody comprises (1) at least 95% or at least 99% of the antibody weight as determined by the Lowry method, (2) using a spinning cup sequenator to determine the N-terminal or internal amino acid sequence. Enough to obtain 15 or more residues, or (3) homogenized by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. Isolated antibody includes the antibody intact in the recombinant cell because at least one member of the antibody's natural environment is not present.

The term “polypeptide” or “protein” refers to a molecule having a sequence of natural protein, ie, a protein that is naturally occurring and specific non-recombinant cells, or that has been genetically engineered or produced by a recombinant cell, Exclusion molecules, molecules in which one or more amino acids of a natural sequence are deleted, added and / or substituted. The terms "polypeptide" and "protein" specifically include sequences in which one or more amino acids of an anti-B7RP1 antibody or anti-B7RP1 antibody have been deleted, added and / or substituted.

The term “polypeptide fragment” means a polypeptide having amino terminal deletions, carboxyl terminal deletions and / or internal deletions. In certain embodiments, the fragments are at least 5 to about 500 amino acids in length. In certain embodiments, the fragment is at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length. Particularly useful polypeptide fragments include functional domains, including binding domains, particularly antigen binding domains, in particular the antigens are epitopes of human B7RP1. In the case of anti-B7RP1 antibodies, useful fragments include, but are not limited to, CDR regions, variable domains of heavy or light chains, portions of antigen chains or variable regions thereof comprising two CDRs, and the like.

The term “specific binding agent” means a naturally occurring or non-naturally occurring molecule that specifically binds a target. Examples of specific binders include, but are not limited to, proteins, peptides, nucleic acids, carbohydrates, and lipids. In certain embodiments, the specific binding agent is an antibody.

"Specific binding agent for B7RP1" refers to a specific binding agent that specifically binds to any portion of B7RP1. In certain embodiments, the specific binding agent for B7RP1 is an antibody that specifically binds B7RP1.

By way of example, when an antibody is labeled, the antibody "specifically binds" to the target if it can compete for its target by the corresponding unlabeled antigen.

As used herein, the term "immunologically functional immunoglobulin fragment" means a polypeptide fragment comprising at least the CDRs of an immunoglobulin heavy and light chain. The immunologically functional immunoglobulin fragments of the invention can bind antigens. In certain embodiments, the antigen is a ligand that specifically binds to a receptor. In this embodiment, the binding of the immunologically functional immunoglobulin fragments of the invention blocks the binding of the ligand to its receptor, thereby blocking the biological response due to binding of the ligand to the receptor. In one embodiment, the immunologically functional immunoglobulin fragments of the invention specifically bind to B7RP1. Preferably, the fragment specifically binds human B7RP1.

The term "naturally occurring" or "natural" as used herein and applied to a subject means that the subject can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including viruses) that can be isolated from a source in nature and not intentionally modified by humans is naturally occurring. As used herein, the term "non-naturally occurring" or "non-naturally" refers to a substance not found in nature or structurally modified or synthesized by humans. For example, "non-naturally occurring" can mean a variant, such as a polynucleotide variant that can be prepared using mutagenesis techniques known in the art or a polypeptide variant produced by the polynucleotide variant. Such variants include those prepared by, for example, nucleotide substitutions, deletions or additions that may involve one or more nucleotides. Polynucleotide variants may be altered in the coding or non-coding regions or both. Alterations in the coding region can provide conservative or non-conservative amino acid substitutions, deletions or additions. Among these are silent substitutions, additions, deletions, and conservative substitutions, which do not alter the properties and activity of the B7RP1 antibodies of the invention. One skilled in the art can readily determine how to generate such variants using methods well known in the art.

The term "operably combined" means that the members to whom the term applies are in a relationship that allows them to perform their own functions under appropriate conditions. For example, a regulatory sequence “operably linked” to a protein coding sequence is bound thereto such that expression of the protein coding sequence can be performed under conditions that are compatible with the transcriptional activity of the regulatory sequence.

As used herein, the term “regulatory sequence” refers to a polynucleotide sequence that can affect the expression, processing or intracellular positioning of operably linked coding sequences. The nature of the regulatory sequence depends on the host organism. In particular embodiments, prokaryotic regulatory sequences may include promoters, ribosomal binding sites, and transcription termination sequences. In other particular embodiments, regulatory sequences of eukaryotes may include promoters, transcriptional enhancer sequences, transcriptional termination sequences and polyadenylation sequences, including one or several recognition sites for transcription factors. In certain embodiments, “regulatory sequences” can include leader sequences and / or fusion partner sequences.

The term "vector" includes a nucleic acid molecule capable of carrying another nucleic acid to which it is bound to a cell. One type of vector is a "plasmid," which refers to a circular double helix DNA loop into which additional DNA segments can be bound. Another type of vector is a viral vector, wherein additional DNA fragments can be bound to the viral genome. Certain vectors can self-replicate in the host cell into which they are introduced (eg, bacterial vectors and episomal mammalian vectors of bacterial replication origin). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the host cell's genome upon introduction into the host cell and thereby replicate with the host genome. Moreover, certain vectors can direct the expression of the genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably since the plasmid is the most widely used vector form. However, the present invention is intended to include providing the same function as such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses).

The phrase “recombinant host cell” (or simply “host cell”) includes cells into which a recombinant expression vector has been introduced. Those skilled in the art will appreciate that the term is intended to mean the progeny of the cell as well as the particular subject cell. Since certain modifications may occur in subsequent generations by mutations or environmental influences, in fact the offspring may not be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. A wide variety of host expression systems can be used to express the antibodies of the invention, including bacteria, yeast, baculovirus and mammalian expression systems (as well as phage display expression systems). An example of a suitable bacterial expression vector is pUC19. To recombinantly express the antibody, the host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody, such that the light and heavy chains are expressed in the host cell so that the host cell It can be secreted in the cultured medium, and the antibody can be recovered from the medium. 2001, MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratories, Ausubel, F.M. (Eds.), Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and Boss, et al. Standard recombinant DNA methodology as described in patent 4,816,397 is used to obtain antibody heavy and light chain genes, incorporate the genes into recombinant expression vectors, and introduce the vectors into host cells.

The term "transduction" refers to the transfer of a gene from one bacterium to another, generally using phage. "Transduction" also refers to the acquisition and delivery of eukaryotic cell sequences by retroviruses.

The term "transfection" refers to the uptake of exogenous or exogenous DNA by a cell, and the cell is "transfected" when the exogenous DNA is introduced into the cell membrane. Many transfection techniques are well known in the art and are disclosed herein. References are made, for example, to Graham et al. 1973, Virology 52: 456; 2001, MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratories of Sambrook et al .; Davis et al., 1986, BASIC METHODS IN MOLECULAR BIOLOGY, Elsevier; and Chu et al. 1981, Gene 13: 197. The technique can be used to introduce one or more exogenous DNA portions into a suitable host cell.

As used herein, the term “transformation” refers to a change in the genetic characteristics in a cell and the cell is transformed when it is modified to include new DNA. For example, when genetically modified in their natural state, the cell is transformed. After transfection or transduction, the transforming DNA can be recombined with DNA derived from the cell by physical integration to the chromosome of the cell, or temporarily maintained as an episomal member without substitution, or as an independent plasmid Can be duplicated with When DNA replicates with cell division, the cell is considered to be stably transformed.

The term “antigen” refers to a molecule or portion of a molecule that can be bound by an optional binder, such as an antibody, and which can additionally be used to produce an antibody in an animal that can bind to the epitope of the antigen. The antigen may have one or more epitopes.

In certain embodiments, antibody variants include glycosylation variants with altered numbers and / or types of glycosylation sites relative to the amino acid sequence of the parent polypeptide. In certain embodiments, protein variants comprise more or fewer N-linked glycosylation sites than natural proteins. The N-linked glycosylation site is characterized by the following sequence: Asn-Xaa-Ser or Asn-Xaa-Thr, wherein the amino acid residues represented by Xaa can be any amino acid residue except proline. Substitution of amino acid residues to create the sequence provides a potential new site for the addition of N-linked carbohydrate chains. Alternatively, substitutions that remove the sequence will remove existing N-linked carbohydrate chains. Rearrangements of N-linked carbohydrate chains are also provided in which one or more N-linked glycosylation sites (typically naturally occurring ones) are removed and one or more new N-binding sites are created. Additional antibody variants include cysteine variants that have one or more cysteine residues deleted or substituted with another amino acid (eg, serine) relative to the parent amino acid sequence. Cysteine variants may be useful when the antibody needs to be folded back into a biologically active batch, such as after separation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than natural proteins and typically have an even number to minimize interactions due to unpaired cysteine.

In further embodiments, antibody variants may include antibodies comprising modified Fc fragments or modified heavy chain constant regions. Fc fragments, meaning "fragment that crystallizes," or heavy chain constant regions, can be modified by mutations to impart altered binding characteristics to the antibody. References are made, for example, to Burton and Woof, 1992, Advances in Immunology 51: 1-84; Ravetch and Bolland, 2001, Annu . Rev. Immunol . 19: 275-90; 2001, Journal of Shields et al. of Biol . Chem 276: 6591-6604; Telleman and Junghans, 2000, Immunology 100: 245-251; Medesan et al. 1998, Eur . J. Immunol . 28: 2092-2100; All of which are incorporated herein by reference). Such mutations include substitutions, additions, deletions, or any combination thereof, and typically include site-directed mutations using one or more mutagenic oligonucleotide (s) according to the methods described herein as well as methods known in the art. (See, e.g., MOLECULAR CLONING: A LABORATORY MANUAL, 3rd Ed., 2001, Cold Spring Harbor, NYand Berger and Kimmel, METHODS IN ENZYMOLOGY, Volume 152, Guide to Molecular Cloning Techniques, 1987, Academic Press, Inc., San Diego, CA., which are incorporated herein by reference).

According to certain embodiments, amino acid substitutions can be made by (1) reduced adaptability to proteolysis, (2) reduced adaptability to oxidation, (3) altered binding affinity, and / or (4) other physicochemical properties of the polypeptide. Or can impart or modify functional properties. According to certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made in naturally occurring sequences (in certain embodiments, portions of a polypeptide outside domain (s) that form intermolecular interactions. in). In certain embodiments, conservative amino acid substitutions typically do not substantially change the structural characteristics of the parent sequence (eg, substituted amino acids tend to collapse or tend to collapse secondary structures that characterize the parent sequence, such as the helix). have). Examples of polypeptide secondary and tertiary structures recognized in the art include PROTEINS, STRUCTURES AND MOLECULAR PRINCIPLES, (Creighton, Ed.), 1984, WH Freeman and Company, New York; INTRODUCTION TO PROTEIN STRUCTURE (C. Branden and J. Tooze, eds.), 1991, Garland Publishing, New York, NY; and Thornton et al., 1991, Nature 354: 105, each of which is incorporated herein by reference.

"Antibody" or "antibody peptide (s)" refers to an intact antibody or a binding fragment thereof that competes with an intact antibody for specific binding. In certain embodiments, binding fragments are provided by recombinant DNA techniques. In further embodiments, the binding fragment is provided by enzymatic or chemical cleavage of the intact antibody. Binding fragments include, but are not limited to, F (ab), F (ab '), F (ab') ₂ , Fv, and single chain antibodies.

The present invention provides antibodies comprising heavy and light chains, wherein the heavy and light chains together form an antigen binding structure capable of specific binding to B7RP1. Full length heavy chains include variable region domains, VH, and three constant region domains, C _H 1, C _H 2, and C _H 3. The VH domain is at the amino terminus of the polypeptide and the C _H 3 domain is at the carboxyl terminus. As used herein, the term “heavy chain” includes full length heavy chains and fragments thereof. Full length light chains contain variable region domains, V _L And constant region domains, C _L. Like the heavy chain, the variable region domain of the light chain is at the amino terminus of the polypeptide. The term "light chain" as used herein includes full length light chains and fragments thereof. The F (ab) fragment consists of one light chain and the variable regions of C _H 1 and heavy chains. The heavy chain of the F (ab) molecule does not form a disulfide bond with another heavy chain molecule. The F (ab ') fragment comprises one light chain and one heavy chain comprising more constant regions between the C _H 1 and C _H 2 domains, so that interchain disulfide bonds can be formed between two heavy chains. (ab ') forms ₂ molecules. The Fv region includes variable regions in both the heavy and light chains, but no constant regions. Single chain antibodies are Fv molecules in which heavy and light chain variable regions are joined by flexible linkers to form a single polypeptide chain, which forms an antigen binding region. Single chain antibodies are discussed in more detail in International Patent Application Publications WO 88/01649 and US Pat. Nos. 4,946,778 and 5,260,203.

Bivalent antibodies other than "multispecific" or "multifunctional" antibodies are understood to include binding sites with antigen specificity that are homologous in certain embodiments.

In the antibody binding and specificity assessment according to the invention, when the excess antibody reduces the amount of ligand bound to the receptor by at least about 20%, 40%, 60%, 80%, 85% or more (especially in vitro In measurement using a competitive binding assay), which substantially inhibits the binding of the ligands to the receptor.

"Neutralizing antibody" means an antibody molecule that can block or substantially reduce the effector function of the target to which it binds. Thus, "neutralizing" anti-B7RP1 antibodies may block or substantially reduce effector functions, such as receptor binding and / or eliciting cellular responses of B7RP1. “Substantially reduced” means at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85% or more of the effector function of the target antigen (eg, human B7RP1), Or a reduction of at least about 90% or more.

The term “epitope” includes any site on an antigen that can specifically bind to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, specific three-dimensional structural features and / or specifics Can have charge characteristics. Epitopes are regions of antigen that are bound by antibodies. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of protein and / or polymer. In certain embodiments, the antibody is the equilibrium dissociation constant of about ^{10 -6 M, 10 -7 M,} 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or about ¹⁰ Less than ¹² M is said to bind specifically to the antigen.

When two antibodies are homologous or recognize an overlapping epitope for spatial placement, they bind to "substantially the same epitope" as the reference antibody. The most widely used and rapid method for determining whether two antibodies bind homologous or overlapping epitopes for spatial placement is a competitive assay, which is placed in all different formats, using labeled antigens or labeled antibodies. Can be. Generally, the antigen is immobilized on the substrate and the ability of the unlabeled antigen to block the labeled antibody is measured using radioisotopes or enzyme labels.

The term "pharmaceutical" is used herein to denote an extract made of a chemical compound, a mixture of chemical compounds, a biological polymer or a biological material.

As used herein, the term “label” or “labeled” refers to a marker detectable by, for example, the incorporation of an amino acid labeled with a radioactive substance or binding to a polypeptide of a biotin moiety that can be detected by labeled avidin. (Eg, streptavidin, chemiluminescent marker or enzymatic activity that can be detected by optical or colorimetric methods, including detectable markers such as fluorescent markers). In certain embodiments, the label can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and can be advantageously used in the methods disclosed herein. Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides such as ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ^{99 m} Tc, ¹¹¹ In, ¹²⁵ I and ¹³¹ I, fluorescence Label (e.g., fluorescein isothiocyanate or FITC, rhodamine or lanthanide phosphor), enzymatic label (e.g. horseradish peroxidase, β-galactosidase, luciferase, alkaline force Patase), chemiluminescent labels, hapten labels such as biotinyl groups, and premeasured polypeptide epitopes recognized by secondary reporters (eg, leucine zipper pair sequences), binding to secondary antibodies Sites, metal binding domains, or epitope tags). In certain embodiments, the label is bound by spacer arms of various lengths (eg, (CH ₂ ) _n , where n <about 20) to reduce potential spatial placement steric hindrance.

The term "biological sample" as used herein includes, but is not limited to, any amount of material from a living individual or from a previously living individual. Such living subjects include, but are not limited to, humans, mice, monkeys, rats, rabbits, and other animals. Such materials include, but are not limited to, blood, serum, urine, cells, organs, tissues, bones, bone marrow, lymph nodes, and skin.

As used herein, the term “pharmaceutical agent or drug” refers to a chemical compound or composition that, when properly administered to a patient, can induce a desired therapeutic effect. The expression “pharmaceutical effective amount” associated with a pharmaceutical composition comprising one or more antibodies of the invention may be used to reverse the sensitivity threshold reduction to restore sensitivity to a level comparable to that observed in healthy subjects in patients according to the invention. It is understood to mean the amount of the pharmaceutical composition which can eliminate the reduction in sensitivity threshold to stimulation.

"Disorder" is any condition in which the treatment according to the invention is of benefit. "Disorders" and "symptoms" are used interchangeably herein and include diseases of the immune system associated with inappropriate immune responses, including pathological conditions that cause predisposition to chronic and acute immune system disorders or disorders that are problematic in mammals. A number of symptoms and disorders for which the treatment according to the invention is of benefit is described, for example, in international patent application PCT / US00 / 01871 (Publication No. WO 00/46240), which is incorporated herein by reference.

The terms “immune disease” and “immune symptom” refer to an increase in B7RP1 levels, any medical condition or disorder associated with increased sensitivity to B7RP1, or autoimmune disease, graft survival, bone marrow, and organ transplantation. , Allograft sensitization due to blood fusion, toxin shock syndrome, T-cell dependent B-cell mediated disease, chronic inflammatory disease associated with chronic immune cell failure, lymphocytic disease (eg, multiple myeloma, Valdestoms macroglobulin) T-cell mediated diseases including Waldenstoms macroglobulinemia and coldoglobulinemias and cancer. Non-limiting examples of autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, immune thrombocytopenic purpura (ITP), multiple sclerosis, diabetes and psoriasis. Non-limiting examples of chronic inflammatory diseases include inflammatory bowel disease (eg, Crohn's disease and ulcerative colitis), Graves disease, Hashimoto's thyroiditis and diabetes.

The terms “immune disease” and “immune symptom” may also include any clinical condition, such as hypersensitivity, alleviated by inhibition of antibody production. Hypersensitivity can be caused, for example, by hay fever, allergy, asthma, atopy and acute edema. Non-limiting examples of diseases that cause antibody mediated hypersensitivity include systemic lupus erythematosus, arthritis (eg, rheumatoid arthritis, semiarthritis, psoriatic arthritis), nephropathy (eg, glomerulonephritis, membranous, glomeruli) Interstitial capillary glomerulonephritis, focal segmentation, focal necrosis, meniscus, and proliferative kidney disease, such as proximal tubular injury, skin disorders (eg, celtic and pseudomolar swelling, nodular erythema), endocrine diseases [eg, thyroiditis, Graves disease (Graves disease, Hashimoto disease, insulin dependent diabetes mellitus], various lung diseases (eg exogenous alveolitis), various angiopathies, celiac disease, diseases caused by abnormal production of IgA, a number of anemia and hypoplateletemia, Guillain Barre syndrome (Guillain-Barre Syndrome) and myasthenia gravis.

As used herein, the terms “effective amount” and “therapeutically effective amount”, when used in connection with a pharmaceutical composition containing a vehicle or one or more anti-human B7RP1 human antibodies, provide a desired result (ie, vehicle, for example). Or an amount sufficient to provide a desired result of therapy with an anti-human B7RP1 human antibody of the invention is a desired regulation of T-cell response) or to support an observable decrease in the level of one or more biological activities of B7RP1, or Refers to the dosage. More specifically, a therapeutically effective amount is intended to inhibit one or more clinically defined pathological processes associated with problematic symptoms, eg, immune disorders and diseases, in a subject treated in vivo with a medicament for a period of time. Sufficient amount of anti-human B7RP1 human antibody (s). In the present invention, an "effective amount" of anti-B7RP1 antibody can modulate T-cell response in a patient. In the methods of the invention, the term "modulation" and its grammatical variants refer to, for example, the prevention, partial or complete inhibition, reduction, delay or slowing down of an immune response. The effective amount may vary depending on the specific vehicle- or anti-human B7RP1 human antibody (s) selected, and also depends on the factors related to the patient to be treated and the severity of the symptoms and disorders. For example, when vehicle- or anti-human B7RP1 human antibody (s) are administered in vivo, dose response line curves and toxicity data obtained in preclinical animal experiments, as well as factors such as the patient's age, weight and health Will be considered. When a medicament comes into contact with in vitro cells, various preclinical in vitro studies will be designed to assess parameters such as absorption, half-life, dosage, toxicity, and the like. Determination of an effective amount or therapeutically effective amount for a given agent is at the discretion of the skilled person.

As used herein, the terms "B7 related protein-1" and "B7RP1" are defined as all mammalian species of natural sequence B7RP1, which is described in International Patent Application Publication WO 00/46240, which is incorporated herein by reference. .

As used herein, “substantially pure” or “substantially purified” means a compound or species that is the predominant species present (ie, richer than any other individual species in the composition on a molar basis). In certain embodiments, the substantially purified fraction is a composition that occupies at least about 50% or more (by molar basis) of all polymeric species present in the species. In certain embodiments, the substantially purified composition comprises greater than about 80%, 85%, 90%, 95% or 99% of all polymeric species present in the composition. In certain embodiments, the species are purified down to essentially homogeneous (contaminant species not detected in the composition by conventional detection methods), wherein the composition consists essentially of a single polymeric species.

The term "patient" includes human and animal subjects.

"Treat" or "treat" refers to both therapeutic treatment and prevention or prevention. Those in need of treatment include those already with the disorder as well as those with the disorder that are susceptible to or have to be prevented.

Unless otherwise indicated in the context, singular terms shall include the plural and plural terms shall include the singular.

According to certain embodiments of the invention, the antibody against B7RP1 may be used to treat immune system disorders and immune system diseases, including but not limited to those mentioned above.

In one aspect of the invention, fully human monoclonal antibodies are provided that have biological and immunological specificities for binding to human B7RP1. In another aspect of the invention, nucleic acids are provided comprising nucleotide sequences encoding amino acid sequences for heavy and light chain immunoglobulin molecules, in particular sequences corresponding to their variable regions. A particular embodiment of this aspect of the invention is the sequence corresponding to the complementarity determining regions (CDRs), in particular CDR1 to CDR3, of the heavy and light chains provided by the invention. In another aspect, the present invention provides hybridoma cells and cell lines expressing antibodies of the invention that are immunoglobulin molecules and antibodies, such as monoclonal. The invention also provides biologically and immunologically purified preparations of monoclonal antibodies with biological and immunological specificities for binding to antibodies such as human B7RP1.

The ability to clone and reconstruct megabase-sized human loci on the yeast artificial chromosome (YAC) and to introduce them into the mouse germline not only uncovers functional members of very large or only mapped loci but also human diseases It provides an advantageous approach to provide a useful model of. Moreover, the use of this technique to replace mouse loci with human equivalents provides unique insight into the expression and regulation of human gene products during development, communication with other systems and their relevance in disease induction and progression.

An important practical application of this strategy is the "humanization" of the mouse humoral immune system. Introduction of the human immunoglobulin (Ig) locus in which the endogenous Ig gene has been inactivated into mice provides an opportunity to study its role in B-cell development as well as to study mechanisms that are rooted in the defined expression and assembly of antibodies. Moreover, this strategy provides a source for the production of fully human monoclonal antibodies (MAbs).

The term “human antibody” includes antibodies with variable and constant regions substantially corresponding to human germline immunoglobulin sequences. In certain embodiments, human antibodies are produced in non-human mammals including, but not limited to, rodents such as mice and rats, and rabbits such as rabbits. In certain embodiments, human antibodies are produced in hybridoma cells. In certain embodiments, human antibodies are produced recombinantly.

The term "recombinant" with respect to an antibody includes an antibody produced, expressed, produced or isolated by recombinant means. Representative examples include animals expressed using recombinant expression vectors transfected into host cells, antibodies isolated from recombinants, combined human antibody libraries, transgenic animals for human immunoglobulin genes (eg, mice) Isolated antibodies (see, for example, Taylor , et al . , 1992, Nucl . Acids Res . 20: 6287-6295); Or antibodies prepared, expressed, generated or isolated by any means involving splicing a human immunoglobulin gene sequence into another DNA sequence. The recombinant human antibody has variable and constant regions derived from human germline immunoglobulin sequences.

Human antibodies have three or more advantages over non-human and chimeric antibodies for use in human therapy: 1) Since the effector portion of the antibody is human, it can further interact with other parts of the human immune system (eg, More efficiently destroy target cells by complementary dependent cytotoxicity (CDC) or antibody dependent cytotoxicity (ADCC); 2) Since the human immune system does not recognize human antibodies as foreign, the antibody response to the injected antibody is less than for non-human antibodies or partially foreign chimeric antibodies that are completely foreign; 3) Injected non-human antibodies have been reported to have a much shorter half-life in the human circulation than the half-life of human antibodies. Infused human antibodies have essentially the same half-life as naturally occurring human antibodies, allowing for fewer and less frequent doses.

Accordingly, fully human antibodies are expected to minimize the immunogenic and allergic responses inherent in mice or mouse induced MAbs, thus increasing the efficacy and safety of the administered antibody. Thus, fully human antibodies of the present invention can be used for the treatment of diseases and disorders associated with inappropriate immune responses, for those requiring repeated antibody administration. Accordingly, one particular advantage of the anti-B7RP1 antibodies of the present invention is that the antibody is a fully human antibody and can be administered non-acute to a patient while being a human anti-mouse antibody or other non-human species described above. Minimize side effects commonly associated with fully human antibodies.

One skilled in the art can engineer mouse strains lacking in mouse antibody preparation with large fragments of the human Ig locus, such that the mice produce human antibodies in the absence of mouse antibodies. Large human Ig fragments can maintain large variable gene diversity as well as proper regulation of antibody production and expression. Due to the utilization of mouse cell organs for antibody diversification and selection and the lack of immunological tolerability for human proteins, the human antibody repertoires produced in these mouse families are highly resistant to any antigen of interest, including human antigens. Provide affinity. Using hybridoma technology, antigen-specific human MAbs with the desired specificity can be produced and selected.

Transgenic animals (eg mice) can also be used to prepare human antibodies in the absence of endogenous immunoglobulin production. For example, moving the human germ cell immunoglobulin gene array to the germ cell mutant mice provides human antibody production upon antigen induction (see, eg, Jakobovits et al., 1993, Proc . Natl . Acad . Sci . USA 90: 2551-2555; 1993, Nature of Jakobovits et al., 362: 255-258; 1993, Year of Bruggemann et al. in Immun . 7:33, 1994, Nature 148: 1547-1553), and 1996, Nature Biotechnology 14: 826; 2000, Nature 404: 995-999 by Gross et al .; And US Patents 5,877,397, 5,874,299, 5,814,318, 5,789,650, 5,770,429, 5,661,016, 5,633,425, 5,625,126, 5,569,825, and 5,545,806, each of which is incorporated herein by reference for all purposes in its entirety. Human antibodies can also be produced in phage display libraries (Hoogenboom and Winter, 1992, J. Mol . Biol 227: 381; Marks et al. 1991, J. Mol . Biol . 222: 581). Techniques such as Cole and Boerner are also Available for the production of human monoclonal antibodies (Cole et al. 1985, MONOCLONAL ANTIBODIES AND CANCER THERAPY Alan R. Liss, p. 77; and Boerner et al. 1991, J. Immunol. 147: 86-95).

Recombinant human antibodies may also be subjected to in vitro mutagenesis (or somatic mutagenesis in vivo when animal transgenics to human Ig sequences are used), thus resulting in amino acids in the V _H and V _L regions of the recombinant antibody. The sequences are derived from those related to human germline V _H and V _L sequences, but in nature may not be present in the human antibody germline repertoire in vivo.

In certain embodiments, one of skill in the art can utilize constant regions derived from species other than human using human variable region (s) in the mouse for chimeric antibody preparation.

Bispecific or bifunctional antibodies are generally artificial hybrid antibodies with two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be provided by a variety of methods including, but not limited to, fusion of hybridomas or binding of F (ab ') fragments. References, For example, Songsivilai & Lachmann, 1990, Clin . Exp Immunol. 79: 315-321; Kostelny et al . 1992, J. Immunol . 148: 1547-1553.

The present invention provides antibodies that bind to human B7RP1. The antibodies can be provided by immunization with full length B7RP1 or fragments thereof. Antibodies of the invention may be polyclonal or monoclonal and / or may be recombinant antibodies. In a preferred embodiment, the antibody of the invention is a human antibody produced by immunization of a transgenic animal capable of producing a human antibody, for example (see, eg, International Patent Application Publication No. WO 0/12227).

The complementarity determining regions (CDRs) of the light and heavy chain variable regions of the anti-B7RP1 antibodies of the invention can be transplanted into framework regions (FR) from the same or another species. In certain embodiments, the CDRs of the light and heavy chain variable regions of the anti-B7RP1 antibody can be transplanted into a consensus human FR. To generate consensus human FRs, FRs derived from various human heavy or light chain amino acid sequences are aligned to identify consensus amino acid sequences. The FRs of the anti-B7RP1 antibody heavy or light chain can be replaced with FRs from different heavy or light chains. Rare amino acids in the FRs of the heavy and light chains of the anti-B7RP1 antibody are generally not replaced and the remainder of the FR amino acids may be replaced. Rare amino acids are specific amino acids where they are generally not found in the FR. The variable region transplanted from the anti-B7RP1 antibody of the invention can be used in a constant region that is different from the constant region of an anti-B7RP1 antibody. Alternatively, the implanted variable region is part of a single chain Fv antibody. CDR transplantation is described, for example, in U.S. Patents 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101, which are incorporated herein by reference for any purpose.

Antibodies of the invention have substantial portions of human antibodies that produce loci inserted into antibody-producing cells of mice, and can be prepared using transgenic mice that have been further engineered to be lacking in the production of endogenous murine antibodies. Such mice can provide human immunoglobulin molecules and antibodies and produce no or substantially reduced weight of murine immunoglobulin molecules and antibodies. The techniques used to achieve these results are disclosed in the patents, applications, and references disclosed herein. In certain embodiments, those skilled in the art can use the methods disclosed in International Patent Application Publication 98/24893, which is incorporated herein by reference for any purpose. References also include Mendez et al., 1997, Nature Genetics 15: 146-156, which is incorporated herein by reference for any purpose.

Monoclonal antibodies (mAb) of the invention can be produced by a variety of techniques including conventional monoclonal methodologies, such as standard somatic hybridization techniques of Kohler and Milstein (1975, Nature 256: 495). Other techniques for producing monoclonal antibodies can be used, such as viral or cancerous transformation of B-lymphocytes.

Exemplary animal systems for hybridoma preparation are mice. Hybridoma production in mice is known in the art and techniques for isolation of immunized splenocytes for immunization protocols and fusions are also known in the art. Fusion partners (eg murine bone marrow cells) and fusion procedures are also known.

In certain embodiments, human monoclonal antibodies against B7RP1 can be provided using transgenic mice bearing parts of the human immune system rather than the mouse system. Such transgenic mice, referred to herein as “HuMab” mice, encode unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences with targeted mutations that will inactivate endogenous μ and γ chain loci. Human immunoglobulin gene minilocus (Lonberg et al., 1994, Nature 368: 856-859). Thus, mice exhibit reduced expression of mouse IgM or κ in response to immunization, and the introduced human heavy and light chain transgenes undergo class alterations and somatic mutations to produce high affinity human IgG κ monoclonal antibodies (Lonberg). Such as The above documents; Lonberg and Huszar, 1995, Intern . Rev. Immunol . 13: 65-93; Harding and Lonberg, 1995, Ann . NY Acad . Sci . 764: 536-546). Preparation of HuMab mice is described by Taylor et al. 1992, Nucleic Acids Res . 20: 6287-6295; Chen et al. 1993, International Immunology 5: 647-656; Tuaillon et al . 1994, J. Immunol . 152: 2912-2920; Lonberg et al. 1994, Nature 368: 856-859; Lonberg, 1994, Handbook of Exp . Pharmacology 113: 49-101; Taylor et al. 1994, International Immunology 6: 579-591; Lonberg & Huszar, 1995, Intern . Rev. Immunol . 13: 65-93; Harding & Lonberg, 1995, Ann . NY Acad . Sci 764: 536-546; Fishwild et al., 1996, Nature Biotechnology 14: 845-851, all of which are incorporated herein by reference. Additional references are disclosed in US Pat. No. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; And 5,770,429; All of these are issued to Lonberg and Kay, and also to US patent no. 5,545,807; International Patent Application Publication WO 93/1227 published June 24, 1993; WO 92/22646 published 12/23/1992; And WO 92/03918, published March 19, 1992, all of which are incorporated herein by reference. Alternatively, the transgenic mouse family described in the Examples below can be used for the production of human anti-B7RP1 antibodies.

The present invention provides human monoclonal antibodies specific for and neutralizing bioactive human B7RP1 polypeptides. In addition, antibody heavy and light chain amino acid sequences are provided that are highly specific for and bind to the B7RP1 polypeptide. This high specificity allows anti-human B7RP1 human antibodies, and human monoclonal antibodies with similar specificities, to be effective immunotherapy against B7RP1 related diseases.

In one aspect, the invention provides an isolated human antibody that binds to the same or essentially the same epitope as the 16H antibody provided herein.

In one aspect, the invention provides isolated humans comprising one or more of the amino acid sequences shown in SEQ ID NOs: 1-40 or 44-58 that bind with a high affinity to a B7RP1 polypeptide epitope and have the ability to antagonize B7RP1 polypeptide activity. Provide an antibody. The antibody may bind to the same or substantially the same epitope as the anti-B7RP1 antibody shown in the Examples herein.

In certain embodiments, the isolated antibody has a dissociation constant (K _D ) of about 10 ^-6 M, 10 ^-7 M, 10 ^-8 M, 10 ^-9 M, 10 ^-10 M, 10 ^-11 M or less binds to B7RP1 polypeptide, inhibits about ^{10 -6 M, 10 -7 M,} 10 -8 M, 10 -9 M or B7RP1 induced survival in an in vitro neutralization test as that of the EC is less than _50. Examples of anti-human B7RP1 human antibodies that meet the aforementioned binding and neutralization criteria are provided herein.

In certain embodiments, anti-human B7RP1 of the present invention are referred to herein as 16H, 16Hg (germline), 5D, 2H, 2Hg (germline), 15H, 41H and 43H. Antibody 16H comprises the V _L and V _H polypeptide sequences as shown in SEQ ID NO: 7 and SEQ ID NO: 1, respectively. Antibody 16Hg comprises a variable light chain (V _L ) and a variable heavy chain (V _H ) as shown in SEQ ID NO: 1 and SEQ ID NO: 8, respectively. Antibody 5D comprises the V _L and V _H polypeptide sequences as shown in SEQ ID NO: 2 and SEQ ID NO: 9, respectively. Antibody 2H comprises the V _L and V _H polypeptide sequences as shown in SEQ ID NO: 3 and SEQ ID NO: 10, respectively. Antibody 2Hg comprises the V _L and V _H polypeptide sequences as shown in SEQ ID NO: 3 and SEQ ID NO: 11, respectively. Antibody 15H comprises the V _L and V _H polypeptide sequences as shown in SEQ ID NO: 4 and SEQ ID NO: 12, respectively. Antibody 41H comprises the V _L and V _H polypeptide sequences as shown in SEQ ID NO: 5 and SEQ ID NO: 13, respectively. Antibody 43H comprises the V _L and V _H polypeptide sequences as shown in SEQ ID NO: 6 and SEQ ID NO: 14, respectively. Features of the anti-human B7RP1 human antibody of the present invention are specifically disclosed in the Examples. Of particular note is the high affinity for the B7RP1 polypeptide and the high ability to antagonize the B7RP1 polypeptide activity demonstrated herein.

시스템(Pharmacia Biosensor, Piscataway, NJ)을 이용한 표면 플라즈몬 공명(SPR)으로 측정한다. 표면 플라즈몬 분석은 또한 분석물 고정(바이오센서 매트릭스 상에 항체) 및 리간드 제시(용액 중 재조합 V)에 의해 수행될 수 있다. 항-인간 B7RP1 인간 항체의 해리 상수(K_D)는 또한 KinExA 방법을 이용하여 결정될 수 있다. 본 발명의 특정 구현예에서, 항체는 약 10^-5 M, 10^-6 M, 10^-7 M, 10^-8 M, 10^-9 M, 10^-10 M, 10^-11 M, 또는 10^-12 M 의 K_D 로 B7RP1 에 결합한다. 용어 "K_D" 는 본원에 사용되는 경우, 특정 항체-항원 상호작용의 해리 상수를 지칭한다. 본 발명의 목적상, K_D 는 하기 실시예에 나타낸 바와 같이 결정했다.The dissociation constant (K _D ) of the anti-human B7RP1 human antibody can be determined by surface plasmon resonance generally described in the Examples below. In general, surface plasmon resonance assays utilize BIAcore to provide real-time interactions between ligands (recombinant B7RP1 polypeptides immobilized on a biosensor matrix) and analytes (antibodies in solution).

Measurement is by surface plasmon resonance (SPR) using a system (Pharmacia Biosensor, Piscataway, NJ). Surface plasmon analysis can also be performed by analyte fixation (antibodies on the biosensor matrix) and ligand presentation (recombinant V in solution). The dissociation constant (K _D ) of the anti-human B7RP1 human antibody can also be determined using the KinExA method. In certain embodiments of the present invention, the antibody is from about ^{10 -5 M, 10 -6 M,} 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 ^-12 Binds to B7RP1 with K _D of M; The term "K _D ", as used herein, refers to the dissociation constant of a particular antibody-antigen interaction. For the purposes of the present invention, K _D was determined as shown in the following examples.

In certain embodiments, the antibody of the invention is of an IgG1, IgG2, IgG3 or IgG4 isotype. The antibody may be of IgG2 or IgG1 isotype. In other embodiments, the antibodies of the invention can be of the IgM, IgA, IgE or IgD isotype. In certain embodiments of the invention, the antibody comprises human kappa light chain and human IgGl, IgG2, IgG3 or IgG4 heavy chain. Expression of an antibody of the invention comprising an IgG1 or IgG2 heavy chain constant region is described in the Examples below. In a particular embodiment, the variable region of the antibody is linked to a constant region other than the constant region for an IgG1, IgG2, IgG3 or IgG4 isotype. In certain embodiments, antibodies of the invention have been cloned for expression in mammalian cells.

In certain embodiments, the conservative modifications (and corresponding modifications to the coding nucleotides) of the heavy and light chains of the anti-B7RP1 antibody are modified to include anti-B7RP1 antibodies with functional and chemical characteristics similar to those of the anti-B7RP1 antibodies disclosed herein. Will provide. In contrast, substantial modifications in the functional and / or chemical characteristics of the anti-B7RP1 antibody may include (a) the structure of the molecular backbone at the substitution region, eg, sheet or helix spacing, (b) molecules at the target site The charge or hydrophobicity of, or (c) its influence on the bulk retention of the side chains may be achieved by selecting substitutions in the amino acid sequences of the heavy and light chains that are significantly different.

For example, a "conservative amino acid substitution" may involve the substitution of an amino acid residue with an unnatural residue that has little or no effect on the polarity or charge of the amino acid residue at that position. Moreover, any natural residue in the polypeptide may also be substituted with alanine as described above as "alanine scanning mutagenesis".

Amino acid substitutions, whether conservative or non-conservative, can be determined by one skilled in the art when such amino acid substitutions are required. In certain embodiments, amino acid substitutions comprise amino acid residues of an anti-B7RP1 antibody (eg, related to the binding specificity and / or affinity of the antibody for B7RP1). Residues involved in binding the antibody to a particular epitope), such as amino acid residues in the CDR1, CDR2 and / or CDR3 regions of the light or heavy chains as described herein. Such amino acid substitutions may increase or decrease the affinity of the anti-B7RP1 antibodies described herein.

Minor changes in the amino acid sequence, such as deletions, additions or substitutions of one, several or several amino acids, can lead to allelic forms of the original protein with substantially identical characteristics. Thus, in addition to the antibodies specifically described herein, other “substantially homologous” antibodies can be readily designed and produced using various recombinant DNA techniques well known to those skilled in the art. In general, modification of the gene can be readily accomplished by a variety of well known techniques, such as site directed mutagenesis. Accordingly, the present invention also contemplates "variant" or "mutant" anti-B7RP1 human antibodies having substantially the same characteristics as the anti-B7RP1 human antibodies disclosed herein (see, for example, WO 00/56772, these All of which are incorporated herein by reference). Accordingly, the term “variant” or “mutant” in connection with an anti-B7RP1 human antibody is any binding molecule (molecule X) that (i) the highly variable regions CDR1, CDR2 and CDR3 of the heavy chain or the highly variable region CDR1 of the light chain. , CDR2 and CDR3 collectively have at least about 80% or more homology, at least about about a highly variable region as shown in SEQ ID NO: 15 to SEQ ID NO: 26 or SEQ ID NO: 27 to SEQ ID NO: 40, respectively At least 90% homology or at least about 95% homology, and (ii) the variant or mutant will inhibit the activity of human B7RP1 to the same extent as the reference anti-B7RP1 human antibody with the same framework region as that of molecule X It means any binding molecule (molecule X) that can be. The antibody may bind to human B7RP1 or mouse B7RP1 or both. Mouse B7RP1 sequences are described in WO 00/46240, which is incorporated herein by reference.

In general, anti-B7RP1 human antibody variants have at least about 80% or more in total for the amino acid sequence represented by SEQ ID NO: 15 to SEQ ID NO: 26 and / or SEQ ID NO: 27 to SEQ ID NO: 40, respectively Amino acid sequence identity, at least about 85% or more sequence identity, at least about 90% or more sequence identity, at least about 91% or more sequence identity, at least about 92% or more sequence identity, at least about 93% or more sequence identity, at least about 94% or more sequence Light and / or heavy chains having identity, at least about 95% or more sequence identity, at least about 96% or more sequence identity, at least about 97% or more sequence identity, at least about 98% or more sequence identity, or at least about 99% or more amino acid sequence identity Have a CDR. The antibody may bind to human B7RP1 or mouse B7RP1 or both.

All anti-B7RP1 human antibody variants have at least about 80% or more amino acid sequence identity, at least about 81% or more sequence identity, at least about 82% or more sequence relative to the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 6 Identity, at least about 83% or more sequence identity, at least about 84% or more sequence identity, at least about 85% or more sequence identity, at least about 86% or more sequence identity, at least about 87% or more sequence identity, at least about 88% or more sequence identity, At least about 89% or more sequence identity, at least about 90% or more sequence identity, at least about 91% or more sequence identity, at least about 92% or more sequence identity, at least about 93% or more sequence identity, at least about 94% or more sequence identity, at least about At least 95% sequence identity, at least about 96% Light chain variable regions having at least about 97% sequence identity, at least about 98% or more sequence identity, at least about 99% or more amino acid sequence identity, and / or all of SEQ ID NOs: 7 to SEQ ID NO: 14 At least about 70% or more amino acid sequence identity, at least about 75% or more sequence identity, at least about 80% or more sequence identity, at least about 81% or more sequence identity, at least about 82% or more sequence identity, at least about 83 At least% sequence identity, at least about 84% or more sequence identity, at least about 85% or more sequence identity, at least about 86% or more sequence identity, at least about 87% or more sequence identity, at least about 88% or more sequence identity, at least about 89% or more Sequence identity, at least about 90% Sequence identity, at least about 91% or more sequence identity, at least about 92% or more sequence identity, at least about 93% or more sequence identity, at least about 94% or more sequence identity, at least about 95% or more sequence identity, at least about 96% or more sequence A heavy chain variable region having identity, at least about 97% or more sequence identity, at least about 98% or more sequence identity, or at least about 99% or more amino acid sequence identity. The antibody may bind to human B7RP1 or mouse B7RP1 or both.

As will be appreciated by those skilled in the art, a number of potential CDR- constant residues can be applied for substitution by other amino acids, allowing the antibody to still maintain substantial affinity for the antigen. Likewise, many framework residues that do not come in contact with CDRs in the heavy and light chains are at a corresponding position from other human antibodies or from other mouse antibodies by human consensus amino acids without substantial loss of affinity or non-immunogenicity of the human antibody. Can accept substitution of amino acids. The selection of various alternative amino acids can be used to provide versions of the disclosed anti-B7RP1 antibodies and fragments thereof with various combinations of affinity, specificity, non-immunogenicity, ease of handling and other desired features.

A “variant” associated with a polynucleotide refers to a nucleic acid molecule having at least about 75% or more nucleic acid sequence identity with a polynucleotide sequence of the present invention. Generally, polynucleotide variants have at least about 75% or more nucleic acid sequence identity, at least about 80% or more nucleic acid sequence identity, at least about 81% or more nucleic acid sequence identity, at least about 82% or more nucleic acid sequence identity with the novel nucleic acid sequences disclosed herein At least about 83% or more nucleic acid sequence identity, at least about 84% or more nucleic acid sequence identity, at least about 85% or more nucleic acid sequence identity, at least about 86% or more nucleic acid sequence identity, at least about 87% or more nucleic acid sequence identity, at least about 88% At least about nucleic acid sequence identity, at least about 89% or more nucleic acid sequence identity, at least about 90% or more nucleic acid sequence identity, at least about 91% or more nucleic acid sequence identity, at least about 92% or more nucleic acid sequence identity, at least about 93% or more nucleic acid sequence identity, enemy At least about 94% or more nucleic acid sequence identity, at least about 95% or more nucleic acid sequence identity, at least about 96% or more nucleic acid sequence identity, at least about 97% or more nucleic acid sequence identity, at least about 98% or more nucleic acid sequence identity, or at least about 99% It has the above nucleic acid sequence identity.

In alternative embodiments, the antibodies of the invention may be expressed in cells other than hybridoma cell lines. In such embodiments, the sequence encoding a particular antibody can be used for transformation of a suitable mammalian host cell. According to this embodiment, the transformation is carried out, for example, by packaging the polynucleotide in a virus (or viral vector) using a virus (or vector) or in a U.S. Any, including transducing host cells by transfection procedures known in the art, as exemplified by patents 4,399,216, 4,912,040, 4,740,461 and 4,959,455 (all of which are incorporated herein by reference for any purpose) Can be achieved using methods of introducing known polynucleotides into host cells. In general, the transformation process used may depend on the host to be transformed. Methods of introducing heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to, dextran mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protozoan fusion, electrophoresis, poly Encapsulation of nucleotide (s) into liposomes and direct microinjection into the nucleus of DNA.

Nucleic acid molecules encoding the amino acid sequences of the heavy chain constant region, heavy chain variable region, light chain constant region or light chain variable region of the anti-B7RP1 antibody of the invention are inserted into a suitable expression vector using standard binding techniques. In one embodiment, the anti-B7RP1 antibody heavy or light chain constant region is appended to the C-terminus of the appropriate variable region and bound to the expression vector. The vector is generally selected to be functional in the particular host cell used (ie, the vector is compatible with host cell organs such that amplification and / or expression of the gene can occur. For an overview of expression vectors, see By METHODS IN ENZYMOLOGY 185 (Goeddel, ed.), 1990, Academic Press.

In general, expression vectors used in any host cell include sequences for plasmid maintenance and cloning and expression of exogenous nucleotide sequences. Such sequences are collectively referred to as "flanking sequences" in certain embodiments, and generally include one or more of the following nucleotide sequences: promoter, one or more enhancer sequences, origin of replication, transcription termination sequence, donor and Complete intron sequence comprising a recipient splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosomal binding site, a polyadenylation site, a polylinker region for nucleic acid insertion encoding a polypeptide to be expressed and a selectable marker member Each of these sequences is discussed below.

Optionally, the vector may comprise an "tag" coding sequence, ie an oligonucleotide molecule located at the 5 'or 3' end of an anti-B7RP1 antibody polypeptide coding sequence; oligonucleotide sequences encoding polyHis (such as hexaHis), or another “tag” such as FLAG, hemaglutinin influenza virus (HA), or myc , in which commercially available antibodies are present. The tag can generally be fused to the polypeptide upon expression of the polypeptide and serve as a means for affinity purification or detection of anti-B7RP1 antibodies from the host cell. Affinity purification can be performed, for example, by column chromatography using antibodies to tags as an affinity matrix. Optionally, the tag can be substantially removed from the anti-B7RP1 antibody polypeptide purified as a variety of methods, such as specific peptidase for cleavage.

The flanking sequences may be homologous (ie, the same species and / or lineage as the host cell), heterologous (ie, species other than the host cell species or lineage), hybrid (ie, combination of flanking sequences from one or more sources), synthetic or It can be natural. As above, the source of flanking sequences can be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that flanking sequences are functional in the host cell organ and are activated by it. It should be possible.

Side sequences useful in the vectors of the invention can be obtained by any method well known in the art. In general, side sequences useful herein have been identified by mapping and / or restriction enzyme cleavage, and thus can be isolated from appropriate tissue sources using appropriate restriction enzymes. In some cases, full length nucleotide sequences of flanking sequences are known. As used herein, flanking sequences can be synthesized using the methods described herein for nucleic acid synthesis or cloning.

컬럼 크로마토그래피(Chatsworth, CA), 또는 기타 당업자에게 공지된 방법을 이용하여 분리함으로써 달성될 수 있다. 상기 목적 달성에 적합한 효소의 선택은 당업자에게 자명하여 용이할 것이다.Regardless of whether all or only some of the flanking sequences are known, polymerase chain reaction (PCR) and / or genomic library screening using suitable probes such as oligonucleotides and / or flanking sequence fragments from the same or another species It can be obtained using. If the flanking sequences are not known, fragments of DNA comprising flanking sequences may be separated from larger pieces of DNA, which may include, for example, coding sequences or another gene or genes. Isolation was followed by restriction digestion to provide suitable DNA fragments followed by agarose gel purification, Qiagen.

Column chromatography (Chatsworth, CA), or by separation using methods known to those skilled in the art. Selection of enzymes suitable for achieving this purpose will be readily apparent to those skilled in the art.

The origin of replication is generally part of commercially available prokaryotic expression vectors, which aid in the amplification of the vector in host cells. If the selected vector does not include the site of origin of replication, it can be chemically synthesized based on known sequences and bound to the vector. For example, the origin of replication from plasmid pBR322 (New England Biolabs, Beverly, Mass.) Is suitable for most Gram-negative bacteria, and has a variety of viral origins [eg, SV40, polyoma, adenovirus, bullous stomatitis. Virus (VSV), or papillomavirus such as HPV or BPV] are useful for vector cloning in mammalian cells. In general, the origin component of replication is not needed for mammalian expression vectors (eg, SV40 origin is often used solely for the purpose of including a viral early promoter).

Transcription termination sequences are generally located 3 'to the terminus of the polypeptide coding region and are provided for transcription termination. Generally, transcription termination sequences in prokaryotic cells include G-C rich fragments followed by poly-T sequences. The sequence is easily cloned from the library or commercially available as part of a vector, which can also be easily synthesized as described herein for methods for nucleic acid synthesis.

Selectable marker genes encode proteins necessary for the survival and growth of host cells growing in selective culture medium. Typical selectable marker genes (a) confer resistance to antibiotics or other toxins such as ampicillin, tetracycline or kanamycin against prokaryotic host cells; (b) compensate for the deficiency of nutritional component of cells; Or (c) codes for a protein that supplies important nutrients that are not available in the complex or diet medium. Exemplary selectable markers are kanamycin resistance gene, ampicillin resistance gene and tetracycline resistance gene. Advantageously, neomycin resistance genes can also be used for selection in both prokaryotic and eukaryotic host cells.

Other selectable genes can be used to amplify the gene to be expressed. Amplification is a process in which genes necessary for the growth or production of proteins important for cell survival are repeated in the later chromosomes of recombinant cells. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and promoter-free thymidine kinase genes. Transformed mammalian cells are placed under selection pressure in which only the transformants are uniquely adapted due to the selectable gene present in the vector. Selective pressure is imposed by culturing the transformed cells under conditions that continuously increase the concentration of the selector in the medium, leading to amplification of antibodies that bind to selectable genes and DNA encoding another gene, such as B7RP1 polypeptide. do. As a result, an expanded polypeptide, such as an anti-B7RP1 antibody, is synthesized from the amplified DNA.

Ribosome binding sites are generally required for initiation of translation of mRNA and are characterized by the Shine-Dalgarno sequence (prokaryote) or the Kozak sequence (eukaryote). Members are generally located 3 'to the promoter of the polypeptide to be expressed and 5' to the coding sequence.

In some cases, for example, when glycosylation is required in eukaryotic host cell expression systems, various pre- or pro-sequences can be handled for glycosylation or yield improvement. For example, it is possible to alter the peptidase cleavage site or add a pro-sequence of a particular single peptide, which can also affect glycosylation. The final protein product may have one or more additional amino acid insertions that are likely to be expressed at the −1 position (based on the first amino acid of the mature protein), which may not be completely eliminated. For example, the final protein product may have one or two amino acid residues found at the peptidase cleavage site, linked to the amino terminus. Alternatively, the use of certain enzyme cleavage sites can provide slightly cleaved forms of the desired polypeptide when the enzyme is cleaved as such in the mature polypeptide.

Expression and cloning vectors of the invention generally comprise a promoter recognized by the host organism and operably linked to a molecule encoding an anti-B7RP1 antibody. The promoter is located upstream (ie 5 ') relative to the start codon of the structural gene (typically about 100 to 1000 bp) that regulates the transcription of the structural gene. Promoters are typically classified into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under certain changes in culture conditions, such as the presence or absence of nutrients or their control corresponding to temperature changes. On the other hand, constitutive promoters constantly transcrib genes to which they are operatively linked, ie have little or no regulation in gene expression. Many promoters that are recognized by a variety of potential host cells are well known. Suitable promoters are operably linked to DNA encoding a heavy or light chain comprising an anti-B7RP1 antibody of the invention by promoter removal from the source DNA by restriction enzyme cleavage and insertion of the desired promoter sequence.

Suitable promoters for use in yeast hosts are well known in the art. Yeast enhancers are advantageously used with yeast promoters. Suitable promoters for use in mammalian host cells are well known and include, but are not limited to, viruses such as polyoma virus, poultry virus, adenovirus (such as adenovirus 2), bovine papilloma virus, chicken breeding virus, cyto Megalovirus, retrovirus, hepatitis B virus and apes virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and actin promoters.

Additional promoters of interest include, but are not limited to, the following: SV40 early promoter (Bernoist and Chambon, 1981, Nature 290: 304-10); CMV promoter (Thomsen et al. 1984, Proc . Natl. Acad . Sci . USA 81: 659-663); Promoters involved in the 3 'long terminal repeat of the Raus sarcoma virus (Yamamoto, et al., 1980, Cell 22: 787-97); Herpes thymidine kinase promoter (Wagner et al., 1981, Proc . Natl . Acad . Sci . USA 78: 1444-45); Promoters and regulatory sequences derived from metallothionine genes (Brinster et al., 1982, Nature 296: 39-42); And prokaryotic promoters, Beta-lactamase promoters (Villa-Kamaroff et al. 1978, Proc . Natl . Acad . Sci USA , 75: 3727-31); Or tac promoter (DeBoer et al., 1983, Proc . Natl . Acad Sci. USA , 80: 21-25). Also of interest are the following animal transcriptional regulatory regions, which exhibit tissue specificity and are used in transgenic animals: the elastase I gene regulatory region active in pancreatic acinar cells (Swift et al. 1984, Cell 38: 639). -46; 1986, Cold by Ornitz et al. Spring Harbor Symp. Quant Biol . 50: 399-409 (1986); MacDonald, 1987, Hepatology 7: 425-515); Insulin gene regulatory regions active in pancreatic beta cells (Hanahan, 1985, Nature 315: 115-22); Immunoglobulin gene regulatory regions active in lymphocyte cells (Grosschedl et al. 1984, Cell 38: 647-58; Adames et al. 1985, Nature 318: 533-38; Alexander et al. 1987, Mol . Cell . Biol . , 7: 1436 -44); Mouse carcinogenic virus regulatory regions active in testis, breast, lymphocytes and mast cells (Leder et al., 1986, Cell 45: 485-95); Albumin gene regulatory regions active in the liver (Pinkert et al. 1987, Genes and Devel . 1: 268-76); Alpha-feto-protein gene regulatory regions active in the liver (Krumlauf et al. 1985, Mol . Cell . Biol . , 5: 1639-48; Hammer et al. 1987, Science 235: 53-58); Alpha 1-antitrypsin gene regulatory region active in the liver (Kelsey et al. 1987, Genes and Devel . 1: 161-71); Beta-globin gene regulatory regions active in bone marrow cells (Mogram et al. 1985, Nature 315: 338-40; Kollias et al. 1986, Cell 46: 89-94); Myelin basic protein gene regulatory region active in oligodendrocytes in the brain (Readhead et al. 1987, Cell 48: 703-12); Myosin light chain-2 gene regulatory region active in skeletal muscle (Sani, 1985, Nature 314: 283-86); And gonadotropin active in the hypothalamus, gonadotropin (Mason et al., 1986, Science 234: 1372-78).

Enhancer sequences can be inserted into the vector by higher eukaryotes to increase the transcription of the DNA encoding the light or heavy chain comprising the anti-B7RP1 antibody of the invention. Enhancers are a length of about 10 to 300 bp, a DNA cis-acting and (cis -acting) member, which acts on the promoter to increase transcription. Enhancers are relatively placement and position independent, found at both 5 'and 3' positions relative to transcriptional units. Several enhancer sequences available in mammalian genes are known (eg, globin, elastase, albumin, alpha-feto-protein and insulin). However, in general, enhancers derived from viruses are used. SV40 enhancers, cytomegalovirus early promoter enhancers, polyoma enhancers, and adenovirus enhancers known in the art are exemplary enhancement members for activation of eukaryotic promoters. Enhancers may be located 5 'or 3' of the coding sequence in the vector, but are generally located at the 5 'site of the promoter.

Expression vectors of the invention can be constructed starting with a vector such as a commercially available vector. The vector may or may not have all desired side sequences. If one or more flanking sequences described herein are not yet present in the vector, they may be obtained separately or linked to the vector. The method used to obtain each flanking sequence is well known to those skilled in the art.

After constructing the vector and inserting the light chain, heavy chain or light chain containing the anti-B7RP1 antibody and the nucleic acid molecule encoding the light chain into the appropriate site of the vector, the finished vector is placed in a suitable host cell for amplification and / or polypeptide expression. Can be inserted. Transformation of the expression vector for the anti-B7RP1 antibody into selected host cells can be transfected, infected, calcium phosphate coprecipitation, electrophoresis, microinjection, lipofection, DEAE-dextran mediated transfection or other known technique. It can be achieved by well known techniques, including. The method chosen will be part of the function of the host cell type to be used. Such methods and other suitable methods are well known to those skilled in the art and are presented, for example, in Sambrook et al., Supra.

Host cells, when cultured under appropriate conditions, can synthesize anti-B7RP1 antibodies and subsequently be collected in culture medium (when host cells secrete it in the medium) or directly from host cells that produce it. (If it is not secreted). Selection of a suitable host cell will depend on various factors such as the desired expression level, polypeptide modifications desired or required for activity (eg glycosylation or phosphorylation) and ease of folding into biologically active molecules.

Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, infant hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocytes Immortalized cell lines available from the American Type Culture Collection (ATCC), including carcinoma cells (eg Hep G2), and many other cell lines. In certain embodiments, the cell line can be selected by determining which cell line has a high expression level and actually produces an antibody with B7RP1 binding properties. In another embodiment, cell lines from a B cell lineage can be selected that do not make their own antibodies but that have the ability to make and secrete heterologous antibodies.

The antibodies of the invention are useful for detecting B7RP1 in biological samples and for identifying cells or tissues that produce B7RP1 protein. Antibodies of the invention that specifically bind to B7RP1 may be useful for the treatment of B7RP1 mediated diseases. The antibody can be used to assay B7RP1 by assaying binding and inhibit B7RP1 from complexing with the B7RP1 receptor. Such antibodies that bind to B7RP1 and block interaction with other binding compounds may have therapeutic use in the regulation of B7RP1 mediated diseases. In certain embodiments, an antibody against B7RP1 may block B7RP1 binding to its receptor, resulting in disruption of the B7RP1 inducible signaling cascade.

The invention also relates to the use of one or more antibodies of the invention in the manufacture of a medicament for the treatment of a disorder or condition caused by increased expression of B7RP1 or increased sensitivity to B7RP1 in a patient, such as the disorder or condition described herein. will be.

In certain embodiments, the present invention provides a pharmaceutical composition containing a therapeutically effective amount of one or a plurality of antibodies of the present invention in combination with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant. to provide. Acceptable formulation materials are nontoxic to receptors at the dosages and concentrations employed. In a preferred embodiment, pharmaceutical compositions containing a therapeutically effective amount of anti-B7RP1 antibody are provided.

In certain embodiments, the material of the acceptable formulation is nontoxic to the receptor at the dosages and concentrations employed.

In certain embodiments, pharmaceutical compositions are formulated to alter, maintain or preserve, for example, the pH, osmotic pressure, viscosity, lightness, color, isotonicity, odor, infertility, stability, dissolution or release, absorption or penetration rate of the composition. It may include a material of. In such embodiments, materials of suitable formulations include, but are not limited to, amino acids (eg, glycine, glutamine, asparagine, arginine or lysine); Antimicrobial agents; Antioxidants (eg, ascorbic acid, sodium sulfite or sodium hydrogen sulfite); Buffers (eg, borate, bicarbonate, tris-HCl, citrate, phosphate or other organic acids); Bulking agents (eg, mannitol or glycine); Chelating agents (eg, ethylenediamine tetraacetic acid (EDTA)); Leveling agents (eg, caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); filler ; Monosaccharides; Disaccharides; And other carbohydrates (eg, glucose, mannose, or dextrins); Proteins (eg, serum albumin, gelatin or immunoglobulins); Coloring, flavoring and diluent; Emulsifiers; Hydrophilic polymers (eg, polyvinylpyrrolidone); Low molecular weight polypeptides; Salt-forming counterions (eg sodium); Preservatives (eg benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorohexidine, sorbic acid or hydrogen peroxide); Solvents (eg glycerin, propylene glycol or polyethylene glycol); Sugar alcohols (eg, mannitol or sorbitol); Suspending agent; Surfactants or wetting agents (eg, pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, tritone, tromethamine, lecithin, cholesterol, tyloxapal); Stability enhancers (eg sucrose or sorbitol); Isotonicity enhancers (eg, alkali metal halides such as sodium or potassium chloride, mannitol sorbitol); Delivery vehicle; Diluent; Excipients and / or pharmaceutical adjuvants. For reference, REMINGTON'S PHAARMACEUTICAL SCIENCES, 18 ^th Edition, (AR Gennaro, ed.), 1990, Mack Publishing Company.

In certain embodiments, the optimal pharmaceutical composition will be determined by one skilled in the art depending, for example, on the intended route of administration, mode of delivery and preferred dosage. References are made, for example, to REMINGTON'S PHAARMACEUTICAL SCIENCES. In certain embodiments, the composition may affect the physical state, stability, rate of in vivo release and rate of in vivo clearance of the antibodies of the invention.

In certain embodiments, the primary vehicle or carrier in the pharmaceutical composition can be aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, physiological saline solution or artificial cerebrospinal fluid, which may be supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin is an additional exemplary vehicle. In certain embodiments, the pharmaceutical compositions of the present invention comprise Tris buffer of about pH 7.0 to 8.5, or acetate buffer of about pH 4.0 to 5.5, and further comprise sorbitol, sucrose, Tween-20 and / or a suitable substitute thereof. It may include. In certain embodiments of the invention, the anti-B7RP1 antibody composition is prepared for storage by mixing the selected composition with the desired purity with an optional formulation agent in the form of a lyophilized cake or aqueous solution (see REMINGTON'S PHAARMACEUTICAL SCIENCES described above). Can be. In addition, in certain embodiments, the anti-B7RP1 antibody product may be formulated as a lyophilisate using suitable excipients such as sucrose.

The pharmaceutical composition of the present invention may be selected for parenteral delivery. Alternatively, the composition may be selected for inhalation or delivery through the digestive tract, such as oral. The preparation of such pharmaceutically acceptable compositions is at the discretion of the skilled person.

Members of the formulation are present at acceptable concentrations at the site of administration. In certain embodiments, the buffer is used to maintain the composition at a physiological pH or slightly lower pH, which is generally in the pH range of about 5 to about 8.

When considering parenteral administration, therapeutic compositions for use in the present invention may be formulated with a parenterally acceptable aqueous solution containing the desired anti-B7RP1 antibody in a pyrogen-free, pharmaceutically acceptable vehicle. It may be provided in the form. Particularly suitable vehicles for parenteral injection are sterile distilled water in which the anti-B7RP1 antibody is formulated in a sterile, properly maintained isotonic solution. In certain embodiments, the formulation may involve the formulation of the desired molecule with a medicament such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic or polyglycolic acid), beads or liposomes, which are depots It may provide a controlled release or sustained release of the product that can be delivered via depot injection. In certain embodiments, hyaluronic acid can also be used, which has the effect of promoting sustained duration in the circulation. In certain embodiments, an implantable drug delivery device can be used for introducing the desired antibody molecule.

The pharmaceutical composition of the present invention may be formulated for inhalation. In this embodiment, the anti-B7RP1 antibody is advantageously formulated as a dry, inhalable powder. In certain embodiments, the anti-B7RP1 antibody inhalation solution can also be formulated with a propellant for aerosol delivery. In certain embodiments, the solution can be sprayed. Pulmonary administration and methods of formulation thereof are also described in international patent application PCT / US94 / 001875, which is incorporated by reference and describes pulmonary delivery of chemically modified proteins.

It is also contemplated that the formulations can be administered orally. Anti-B7RP1 antibodies administered in this manner may be formulated in the presence or absence of a carrier commonly used in the formulation of solid dosage forms such as tablets and capsules. In certain embodiments, the capsule may be designed to release the active portion of the formulation at a point where bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to promote uptake of anti-B7RP1 antibodies. Diluents, flavors, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents and binders may also be used.

Pharmaceutical compositions of the invention are provided so as to contain an effective amount of one or multiple anti-B7RP1 antibodies in a mixture with nontoxic excipients suitable for the manufacture of tablets. By dissolving the tablets in sterile water or other suitable vehicle, the solution can be prepared in unit dosage form. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; Or binders such as starch, gelatin or acacia; Or lubricants such as magnesium stearate, stearic acid or talc and the like.

Additional pharmaceutical compositions will be apparent to those skilled in the art, including formulations involving anti-B7RP1 antibodies in sustained-release or control-delivery formulations. Various other sustained-release or control-delivery means such as liposome carriers, biodegradable particulates or porous meads and depot injection formulation techniques are also known to those skilled in the art. Reference is, for example, international patent application PCT / US93 / 00829, which is incorporated by reference and describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. Sustained release formulations may include semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. Sustained release matrices include polyesters, hydrogels, polyactides (described in US Pat. No. 3,773,919 and European Patent Application Publication EP 058481, each of which is incorporated by reference), L-glutamic acid and gamma ethyl-L-glutamate Copolymers (Sidman et al. 1983, Biopolymers 22: 547-556), poly (2-hydroxyethyl-methacrylate) (Langer et al. 1981, J. Biomed . Mater . Res . 15: 167-277 and Langer , 1982, Chem . Tech . 12: 98-105), ethylene vinyl acetate (supra from Langer et al . ) Or poly-D (-)-3-hydroxybutyric acid (European Patent Application Publication EP 133,988). Sustained release compositions may also include liposomes, which may be prepared by various methods known to those skilled in the art. References are made, for example, to Eppstein et al. 1985, Proc . Natl . Acad . Sci . USA 82 : 3688-3692; European Patent Application Publication EP 036,676; EP 088,046 and EP 143,949, all of which are incorporated by reference.

Pharmaceutical compositions for in vivo administration are generally provided as sterile preparations. Sterilization can be accomplished by filtration through sterile filtration membranes. If the composition is lyophilized, sterilization using the method may be performed before or after lyophilization and reconstruction. Compositions for parenteral administration may be stored in lyophilized form or in solution. Parenteral compositions are generally placed in a container with a sterile access port, for example a reservoir with an intravenous solution bag or a vial with a subcutaneous needle stopper.

Once the pharmaceutical composition is formulated, it can be stored as a solution, suspension, gel, emulsion, solid or as a dehydrated or lyophilized powder. The formulations can be stored in the form used on the fly or in a form which is rebuilt before administration (eg lyophilized).

The invention also provides kits for the preparation of single dose dosage units. Kits of the present invention may comprise both a first container with dried protein and a second container with an aqueous formulation. In certain embodiments of the invention, kits are provided comprising single and multi-space pre-filled syringes (eg, liquid injections and lyosyringe).

An effective amount for use in the treatment of an anti-B7RP1 antibody-containing pharmaceutical composition depends, for example, on the therapeutic flow and the subject. Those skilled in the art will appreciate that appropriate dosage levels for treatment include, in part, the molecule to be delivered, the indication that an anti-B7RP1 antibody is used, the route of administration and the patient's numerical size (body weight, body surface area or organ size) and / or condition ( Age and general health). In certain embodiments, the clinician may titrate the dosage and alter the route of administration to obtain the optimal therapeutic effect. Typical dosages may range from about 0.1 μg / kg up to about 30 μg / kg or more, depending on the factors mentioned above. In certain embodiments, the dosage is 0.1 μg / kg to about 30 μg / kg; 1 μg / kg to about 30 μg / kg; Or 5 μg / kg to about 30 μg / kg.

The frequency of administration will depend on the pharmacokinetic parameters of the particular anti-B7RP1 antibody in the formulation used. Typically, the clinician administers the composition until the dosage achieves the desired effect. Thus, the compositions may be administered over time in a single dose or in two or more doses (which may or may not include the same molecule of interest) or may be administered in continuous infusion via an implantation device or catheter. Further requalification of appropriate dosages is routinely performed by those skilled in the art and is the area of work performed routinely by them. Appropriate dosages can be ascertained by the use of appropriate dose-response data. In certain embodiments, antibodies of the invention can be administered to a patient over an extended period of time. Chronic administration of the antibodies of the present invention minimizes the general adverse immune or allergic reactions associated with antibodies directed against human antibodies in non-human animals, eg, non-complete human antibodies produced in non-human species.

The route of administration of the pharmaceutical composition may be, for example, by injection by oral, intravenous, intraperitoneal, intracerebroventricular (endothelium), intraventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional routes; According to the known method via a sustained release system or an implantation device. In certain embodiments, the compositions can be administered by bolus injection or by continuous infusion or by implantation device.

The composition can also be administered topically via implantation of a membrane, sponge or other suitable material into which the desired molecule is absorbed or encapsulated. In certain embodiments, where an implantation device is used, the device may be implanted in any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, time-release bolus or continuous administration.

In vitro administration of the anti-B7RP1 antibody pharmaceutical composition according to the invention may also be desirable. In such cases, cells, tissues, or organs removed from the patient are exposed to the anti-B7RP1 antibody pharmaceutical composition, which is subsequently transplanted back to the patient.

In particular, anti-B7RP1 antibodies can be delivered by transplanting certain cells genetically engineered using methods such as those described herein to express and secrete polypeptides. In certain embodiments, the cells are animal or human cells and may be autologous, heterologous, or heterologous. In certain embodiments, the cells can be immortalized. In other embodiments, cells can be encapsulated to avoid the invasion of surrounding tissue to reduce the chance of an immunological response. In further embodiments, the encapsulating material is generally a biocompatible, semipermeable polymeric envelope or membrane that allows release of the protein product (s) but prevents destruction of cells by the patient's immune system or other detrimental factors into surrounding tissue.

The following examples, including the experiments performed and the results achieved, are provided for illustrative purposes only and are not intended to limit the invention.

Example  One

Human against B7 related protein-1 (B7RP1) Monoclonal   Preparation of Antibodies

antigen

CHO cells transfected to express purified recombinant human B7RP-1 (hB7RP-1), or hB7RP-1, prepared as described in International Patent Application Publication WO 00/46240, which is incorporated herein by reference (ECACC agent). 85050302) was used as the antigen. Mature human B7RP-1 has the amino acid sequence of residues X to 302 in which X may be 19, 20, 21, 22, 24 or 28 in the sequence represented by SEQ ID NO: 17 in WO 00/46240.

Transgenic HuMab   mouse

Fully human monoclonal antibodies against B7RP-1 were prepared using the HCo7 and HCo12 strains of HuMab transgenic mice expressing human antibody genes. In these two mice, the endogenous mouse kappa light chain gene was disrupted homogenously as described in Chen et al. (1993) EMBO J. 12: 811-820, and the endogenous mouse heavy chain gene was disclosed in PCT publication WO 01/09187. Destroyed homogeneously as described in Example 1 Each of these mouse strains is described by Fishwild et al. (1996) Nature Human kappa light chain transgene, KCo5, as described in Biotechnology 14: 845-851. HCo7 strains are described in US Pat. No. 5,545,806; 5,625,825; And HCo7 human heavy chain transgene as described in 5,545,807. The HCo12 strain carries the HCo12 human heavy chain transgene as described in Example 2 of PCT Publication WO 01/09187.

HuMab immunization :

To provide fully human monoclonal antibodies against B7RP-1, HuMab mice of HCo7 or HCo12 strain were immunized with purified recombinant B7RP-1 or CHO cells transfected to express B7RP-1. General immunization schemes for HuMab mice are described in Lonberg et al. (1994 ) Nature 368 (6474): 856-859; Fishwild Et al. (1996) Nature Biotechnology 14: 845-851 and PCT publication WO 98/24884. Mice were 6-16 weeks of age upon the first injection of antigen. Purified recombinant preparations of B7RP-1 antigen (50 μg) or preparations of transfected CHO cells (3.5 × 10 ⁶ to 1 × 10 ⁷ cells) were used to immunize HuMab mice intraperitoneally.

Transgenic mice were immunized twice intraperitoneally with purified antigen in complete Freund's adjuvant, followed by 2-4 weeks of IP immunization (up to 8 total immunizations) with purified antigen in incomplete Freund's adjuvant. . Immunization with CHO cells transfected to express B7RP-1 was the same except that no full Freund's adjuvant and incomplete Freund's adjuvant were used in the cells. Immune responses were monitored by orbital posterior blood collection. Plasma was screened by ELISA (as described below) and mice with sufficient titers of anti-B7RP-1 human immunoglobulin were used for fusion. Mice were boosted intravenously with antigen 3 and 2 days before sacrifice and spleens removed. In general, 10 to 20 fusions were performed for each antigen. Several mice were immunized for each antigen. A total of 28 HCo7 and HCo12 mouse families were immunized with B7RP-1.

To produce anti-B7RP-1 antibodies HuMab   Screening of Mice:

To screen for HuMab producing antibodies bound to B7RP-1, serum from immunized mice was tested by ELISA as described by Fishwild et al. (1996). Briefly, microtiter plates were coated with 50 μl / well of purified recombinant B7RP-1 at 1-2 μg / ml in PBS, incubated overnight at 4 ° C., and then 5% chicken in PBS / Tween (0.05%). The serum was blocked with 200 μl / well. Dilutions of serum from B7RP-1-immunized mice were added to each well and incubated for 1-2 hours at ambient temperature. Plates were washed with PBS / Tween and then treated with goat-anti-human IgG Fc polyclonal antibody conjugated with horseradish peroxidase (HRP) for 1 hour at room temperature. After washing, plates were developed with ABTS substrate (Sigma, A-1888, 0.22 mg / ml) and analyzed spectrophotometrically at OD 415-495. Mice developed with the highest titer of anti-B7RP-1 antibody were used for fusion. Fusion was performed as described below, and hybridoma supernatants were tested for anti-B7RP-1 activity by ELISA.

Generation of hybridomas producing human monoclonal antibodies to B7RP-1:

Mouse splenocytes isolated from HuMab mice were fused to mouse bone marrow cell lines using PEG based on standard protocols. The hybridomas obtained were then screened for the generation of antigen-specific antibodies. Single cell suspensions of splenic lymphocytes from immunized mice were fused with one quarter of SP2 / 0 unsecreted mouse bone marrow cells (ATCC CRL 1581) population with 50% PEG (Sigma). Cells were plated at about 1 × 10 ⁵ / well in plate bottom microtiter plates, followed by 10% fetal calf serum, 10% P388D1 (ATCC, CRL TIB-63) conditioned medium, 3-5% IGEN in DMEM (Mediatech) , CRL 10013, large amounts of glucose, L-glutamine and sodium pyruvate) and 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 mg / ml gentamycin and 1x HAT (Sigma, CRL P-7185) Incubate for about 2 weeks in the medium. After 1-2 weeks, the cells were cultured in a medium in which the HAT was substituted with HT. Individual wells were then screened by ELISA (as described above) against human anti-B7RP-1 monoclonal IgG antibodies. Once intensive hybridoma growth occurred, medium was monitored after 10-14 days. Antibody-secreting antibodies were replated, screened again, and if still positive for human IgG, diclosure was limited to subcloning the anti-B7RP-1 monoclonal antibody two or more times. Stable subclones were then cultured in vitro to provide small amounts of antibody in tissue culture medium for further characterization.

Example  2

Anti-B7RP1 antibody Heavy chain   And Light chain Cloning

시약(Invitrogen)을 이용하여 전체 RNA 를 분리하기 위한 공급원으로서 이용했다. 5' RACE(cDNA 말단의 신속한 증폭) 올리고뉴클레오티드(5'-CGA CUG GAG CAC GAG GAC ACU GAC AUG GAC UGA AGG AGU AGA AA-3' ; SEQ ID NO : 69)를 GeneRacer^TMKit (Invitrogen) 구성원들 및 프로토콜을 이용하여 RNA 에 결합시켰다. 첫번째 가닥 cDNA 는 신장 어댑터(5'-GGC CGG ATA GGC CTC CAN NNN NNT-3') (SEQ ID NO : 59)을 사용한 랜덤 프라이머를 이용하여 합성하고, 5' RACE(cDNA 말단의 신속한 증폭) 예비 검정을 제조사의 지시사항에 따라 GeneRacer^TM Kit(Invitrogen)을 이용하여 수행했다. cDNA 를 코드하는 완전한 경쇄 제작을 위해, 전방향 프라이머는 GeneRacer^TM 에 있는 프라이머였고, 역방향 프라이머는(5'-GGG GTC AGG CTG GAA CTG AGG-3'(SEQ ID NO : 60)였다. 중쇄의 가변 영역을 코드하는 cDNA 제조를 위해, 전방향 프라이머는 GeneRacer^TM 에 있는 프라이머였고, 역방향 프라이머는 (5'-TGA GGA CGC TGA CCA CAC G-3') (SEQ ID NO : 61)였다. RACE 생성물은 pCR4-TOPO(Invitrogen Corporation)에 클로닝하고, 서열을 결정했다. 콘센서스 서열은 전장 항체 사슬 PCR 증폭을 위한 프라이머 고안에 이용했다.Hybridomas expressing the B7RP1 binding monoclonal antibody 16H are known as TRIzol.

Reagent (Invitrogen) was used as a source for separating total RNA. 5 'RACE (fast amplification of cDNA terminus) oligonucleotides (5'-CGA CUG GAG CAC GAG GAC ACU GAC AUG GAC UGA AGG AGU AGA AA-3'; SEQ ID NO: 69) to GeneRacer ^™ Kit (Invitrogen) members And bound to RNA using protocol. First strand cDNA was synthesized using random primers using an extension adapter (5'-GGC CGG ATA GGC CTC CAN NNN NNT-3 ') (SEQ ID NO: 59) and preliminary 5' RACE (quick amplification of cDNA ends) Assays were performed using the GeneRacer ^™ Kit (Invitrogen) according to the manufacturer's instructions. For complete light chain construction encoding the cDNA, the forward primer was the primer in GeneRacer ^™ and the reverse primer was (5′-GGG GTC AGG CTG GAA CTG AGG-3 ′ (SEQ ID NO: 60). For cDNA preparation encoding the region, the forward primer was the primer in GeneRacer ^™ and the reverse primer was (5′-TGA GGA CGC TGA CCA CAC G-3 ′) (SEQ ID NO: 61). It was cloned into pCR4-TOPO (Invitrogen Corporation) and the sequence was determined The consensus sequence was used to design primers for full length antibody chain PCR amplification.

For cDNA preparation encoding the anti-B7RP1 16H kapp light chain, the 5 'PCR primers were prepared at the amino terminus of the signal sequence, the Xba I restriction enzyme site, and the optimized Kozak sequence (5'-CAG CAG AAG CTT CTA GAC CAC CAT GGA CAT Coded GAG GGT CCT CGC TCA GCT CCT GGG-3 ') (SEQ ID NO: 62). The 3 'primers coded Sal I restriction enzyme sites (5'-CTT GTC GAC TCA ACA CTC TCC CCT GTT GAA GCT C-3') (SEQ ID NO: 63) as well as the carboxyl terminal and termination codons. The resulting PCR product fragment was purified, digested with Xba I and Sal I, gel separated and bound to the mammalian expression vector pDSRα20 (reference is to International Application Publication WO 90/14363, which is hereby incorporated for any purpose herein). PDSRα20 was prepared by modifying nucleotide 2563 at pDSRα19 from “guanosine” to “adenosine” by site directed mutagenesis). The pDSRα19 was prepared by modifying pDSRα2 having the following structure: a sequence containing a transcription termination / polyadenylation signal from the alpha subunit of bovine pituitary glycoprotein hormone alpha-FSH (follicle stimulating hormone) Was modified to shorten to approximately 1400 base pairs in length from the end of the NdeI site to 885 base pairs; The dihydrofolate reductase (DHFR) promoter containing 209 base pairs was shortened by approximately 1 kilobase at the 5 'end; Approximately 550 base pair BglII fragments were deleted from the DHFR polyA sequence.

To prepare cDNA encoding the anti-B7RP1 16H heavy chain, the 5 'PCR primers were prepared using the amino terminus of the signal sequence, the Xba I restriction site, the optimized Kozak sequence (5'-ACA ACA AAG CTT GTA CAC CAT GGA GTT GGG). Coded GCT GAA CTG G-3 ') (SEQ ID NO: 64). The 3 'primer encoded the carboxyl terminus of the variable region comprising the naturally occurring sense strand BsmB I site (5'-GTG GAG GCA CTA GAG ACG GTG ACC AGG ATT CC-3'; SEQ ID NO: 65). To purify the obtained product, Cleavage with Xba I and BsmB I, gel separation and binding to pDSRα20 vector comprising human IgG1 constant region and pDSRα20 vector comprising human IgG2 constant region. All hybridoma inducible anti-B7RP1 heavy chain variable regions, whether natural or not, were cloned as described above in a pDSRα20 vector comprising human IgG1 and human IgG2 constant regions.

Example  3

Chinese hamster ovary ( CHO ) Expression of anti-B7RP1 antibody in cells

Stable expression of the 16H anti-B7RP1 mAb was achieved using the calcium phosphate method (full length 16H heavy chain is shown in SEQ ID NO: 44; 16H kappa chain sequence is shown in SEQ ID NO: 45) 16H-heavy chain / pDSRα19 IgG2 B7RP1 -kappa / pDSRα19 plasmid was achieved by cotransfection into dihydrofolate reductase deficiency (DHFR ⁻ ) serum ^- free adaptive Chinese hamster ovary (CHO) cells. Transfected cells were selected in media containing dialysis serum but hypoxanthine-thymidine to ensure the growth of cells expressing DHFR enzyme. Transfected clones were screened using assays such as ELISA to detect expression of 16H anti-B7RP1 mAb in conditioned media. The best expressing clones were applied to increasing concentrations of methotrexate (MTX) for DHFR amplification. MTX amplified clones were screened using assays such as ELISA to detect higher expression of 16H anti-B7RP1 mAb in conditioned media. The highest expressing clones were subjected to subcloning for uniform population yield and cell bank creation.

Other recombinant anti-B7RP1 antibodies of the invention can be generated in DHFR deficient Chinese hamster ovary cells using the protocols described above for anti-B7RP1 monoclonal antibodies. The DNA sequence encoding the complete heavy or light chain of each anti-B7RP1 antibody of the invention is cloned into an expression vector. CHOd-cells (ATCC CRL-9096 or ECACC No. 94060607) were cotransfected with an expression vector capable of expressing a complete heavy chain and an expression vector expressing a complete light chain of a suitable anti-B7RP1 antibody. For example, for the production of 5D anti-B7RP1 antibodies, a cell comprises a vector capable of expressing a complete heavy chain comprising the amino acid sequence shown in SEQ ID NO: 47 and an amino acid sequence shown in SEQ ID NO: 48. Cotransfected with a vector capable of expressing the complete light chain. Table 2 summarizes exemplary complete light chains and exemplary complete heavy chains for anti-B7RP1 antibodies with human IgG heavy chain constant regions. Those skilled in the art will appreciate that IgG1 or IgG2 may be substituted for one another (ie where IgG1 is listed in the table, IgG2 may be present and vice versa). Alternatively, any other immunoglobulin (eg, IgM, IgA, IgE or IgH) can be used to produce the antibodies of the invention.

Example  4

Production of Anti-B7RP1 Antibodies

Anti-B7RP1 antibodies are produced by expression in clonal lines of CHO cells. At each production run, cells from a single vial are thawed in serum-free cell culture medium. Cells are first grown in a T-flask and then scaled up to a spinner flask, followed by a 2000 L bioreactor, in a stainless steel reactor. Production is carried out in a 2000 L bioreactor using fed-batch cultures, in which a nutrient infusion comprising concentrated media members is added to maintain cell growth and culture viability. Anti-B7RP1 antibodies are produced structurally by cells and continue production for about 2 weeks when secreted into cell culture medium.

The production reactor is controlled at predetermined pH, temperature and dissolved oxygen levels: pH is controlled by addition of carbon dioxide gas and sodium carbonate; Dissolved oxygen is controlled by air, nitrogen and oxygen gas flows.

At the end of production, the cell broth is injected into a disc stack centrifuge and the cell supernatant is separated from the cells. The concentrate is clarified through a depth filtration filter followed by a 0.2 μm filter. The clarified conditioned medium is then concentrated by tangential ultrafiltration. Conditioned medium is concentrated 15- to 30-fold. The concentrated conditioned medium obtained is then processed via purification or frozen for later purification.

Example  5

16H mAb   Reproductive system

Sequence alignment of the 16H antibody with human germline sequences showed that the framework sequences of the variable regions of the 16H antibody were nearly identical, with only three amino acids different from the V _H 3-07 and JH4 germline sequences ( FIG. 1A ). . The framework sequence for the VK region of the 16H antibody was found to be identical to the VK1-L15 germline sequence. Excessive mutations in somatic cells are theoretically possible to be recognized as foreign in the patient's immune response; In this case, the patient exhibits an anti-idiotypic response that neutralizes the treatment. To reduce the potential, V _H frame caught up on the 3 amino acid changes in the work area as a V _H 3-07 and JH4 germline sequences (Figure 1A). Since germline V _H and J _H gene segments are present in all human genomes, germline versions of 16H are not recognized as foreign by the immune response of the administered patient. Bio black plate co-stimulation is a non-reproductive antibodies gyeyeolwa - was performed to determine whether it is possible to induce a T- cell proliferation with a similar IC ₅₀ and IC ₅₀ of the integration of reproductive antibodies. Costimulatory assays were performed as described below using anti-CD3 and hB7RP-1-Fc fusion proteins and confirmed that said germline antibody, referred to as 16H germline or 16Hg, retains its biological activity ( FIG. 1B). ).

Example  6

Biacore ® and KinExA  On by Monoclonal   Affinity Determination of Antibodies

Three antibodies (5D and 16H were prepared as described in Example 1, and 16H germline were prepared as described in Example 5) were purified and subjected to binding affinity analysis. B7RP1-Fc was fixed at high density on the CM5 sensor chip using standard amine coupling chemistry. The fixed concentrates of mAbs were then incubated at room temperature for at least 8 hours with varying concentrations of B7RP-1 or B7RP1-Fc so that they reached equilibrium. The sample was then injected onto the B7RP1-Fc surface and the binding signal observed showed the free antibody remaining in solution at equilibrium. Using two different antibody concentrations (0.2 nM and 1 nM), the K _D of the interaction between a specific mAb and ligand is a competition curve using a dual-curve one-site homogeneous binding model. Nonlinear regression analysis (Adamczyk et al. 1999, Bioconjugate). Chem . 10: 1032-37; 2000, Methods 20: 319-28, Adamczyk et al. As shown in FIG. 2 and Table 3, 16H, 16Hg and 5D mAbs all bound with high affinity to both soluble B7RP-1 and B7RP-1-Fc proteins. In addition, the results suggest that 16H (non-germline) and 16Hg (germline) respond similarly, demonstrating that germ lineage does not significantly affect binding between antibody and ligand.

Binding of 5D, 2H and 2H germline antibodies was also tested using KinExA (kinetic exclusion assay) technology. In this assay, hB7RP-1 was coupled to agarose beads. Beads were used to provide bead columns. Samples containing antibodies at fixed concentrations were allowed to equilibrate at different concentrations of hB7RP-1, followed by a bead column. Antibodies that do not complex with the ligand bind to the coated beads.

Fluorescently tagged anti-human Fc secondary antibodies were used to detect bound test antibodies. The signal obtained at a given ligand concentration was proportional to the free antibody in solution. Using two different antibody concentrations, K _D of the interaction was calculated from nonlinear regression analysis of the competition curve using a double curve single site homogeneous binding model (Adamczyk et al. 1999, Bioconjugate Chem. 10: 1032-37; Adamczyk et al. 2000, Methods 20: 319-28). 3, 4 and 5 show double curve pits for antibodies 5D, 2H and 2H (germline). Using this technique, about 10-fold differences in K _D s for antibodies 5D and 2H were seen.

및 KinExA 검정의 결과는 항체 5D 가 2H 또는 16H 보다 hB7RP-1 에 대한 더 높은 친화성을 가졌음을 증명했다. 또한, 항체 2H 의 생식계열 버젼은 비-생식계열 구축물과 현저한 차이를 나타내지 않는다.Biacore

And the results of the KinExA assay demonstrated that antibody 5D had a higher affinity for hB7RP-1 than 2H or 16H. In addition, the germline version of antibody 2H does not show significant differences from non-germline constructs.

Example  7

Functional Features of Anti-B7RP1 Antibodies

Functional features of the B7RP-1 antibodies of the present invention were evaluated using a binding competition assay, an in vitro costimulatory assay and an in vitro tetanus toxoid assay.

Combined competition study

Binding competition studies were performed using 16H mAbs to demonstrate that they can compete for ICOS binding to B7RP-1. CHO cells transfected with a gene encoding full-length human B7RP-1 were first incubated with the weighted unlabeled 16H mAb and subsequently stained with the fluorescently labeled ICOS-Fc fusion protein. The cells were then analyzed using flow cytometry. As shown in FIG. 6, ICOS-Fc stains B7RP-1-transfected CHO cells; 0.4 μg / ml of 16H mAb does not affect ICOS-Fc binding. However, 6 and 25 μg / ml of 16H competed effectively with ICOS-Fc binding, suggesting that 16H mAb competes for ICOS on B7RP-1.

Co -Stimulation black

Cell culture plates (Falcon, Cat No. 353077, U bottom) were prepared using 1 μg / ml anti-human CD3 antibody (PharMingen Cat No.555336) and 10 μg / ml anti-human IgG (Fc specific Sigma Cat No.I3391). Was coated using. Anti-CD3 antibody and anti-human immunoglobulin in phosphate buffered saline (PBS) were added to each well (100 μl / well). The coated plate was incubated overnight at 4 ° C. or for 2 hours at room temperature. The plate was then washed twice with PBS. After washing, 1 μg / ml human B7-2Fc (R & D System, Cat No. 141-B2) or 5 μg / ml hB7RP1Fc, respectively, was diluted in PBS and added to each well (100 μl per well). The plate was then incubated at room temperature for 3 hours and then washed twice with PBS. Purified human T cells were treated with 200 μl volume of medium (10% fetal bovine serum (FCS), penicillin-streptomycin-L-glutamine (PSG), β-mercaptoethanol (2-ME), N-acetyl aspartate. (NAA) and RPMI 1640 supplemented with Napyruvate) (1 × 10 ⁵ per well) and incubated for 48 hours at 37 ° C., 5% CO ₂ . ^3- H thymidine (ICN Cat No. 2404205) was added at 1 μCi / well and cells were incubated overnight at 37 ° C. 5% CO ₂ . The cells were then harvested and counted.

Cell culture plates (Falcon, Cat No. 353077, U bottoms) were coated as above with 0.1 μg / ml anti-human CD3. hB7RP1 transfected CHO cells (5000RAD irradiation) were added at 2 × 10 ⁴ per well, then purified human T cells were added at a volume of 200 μl at 1 × 10 ⁵ per well. The plate was incubated as above at 37 ° C., 5% CO ₂ for 48 hours. ^3- H thymidine was added at 1 μCi / well. Cells were incubated overnight as above, pooled and counted.

tetanus Toxoid   black

PBMC was purified from human blood using the Ficoll-Paque (Amersham Biosciences) gradient as follows. Blood is diluted 1: 2 using PBS, the diluted blood layered on top of Ficoll (1/3 room temperature Ficoll + 2/3 diluted blood) and centrifuged at room temperature for 30 minutes at 2500 rpm. The upper layer (serum and platelets) was then aspirated off and the mononuclear cell layer was transferred to a new 50 ml tube. The isolated PBMCs were diluted with PBS (three times volume of mononuclear cell layer), centrifuged at 1300 rpm for 30 minutes at room temperature and washed as above. PBMC was resuspended in medium (RPMI 1640 + 10% heat inactivated FBS + 1 × PSG + 1 × NEAA + 55 μM 2-ME) and cells were counted.

PMBC was added to the wells of a 96-well round bottom plate at 100 ml PBMC / well (3 × 10 ⁶ / ml). Tetanus toxoid (20 μg / ml; University of Massachusetts) was added to a final concentration of 5 μg / ml. Cells were incubated at 37 ° C. for 3 days; 100 μl of supernatant was collected and incubated for an additional 6-8 hours in the presence of 1 μCi / well ³ H-thymidine (MP Biomedicals). The cells were then harvested and counted.

Table 4 summarizes the functional characteristics of certain antibodies of the invention as determined using the assay described above.

Example  8

Epitope Mapping

Experiments were performed to identify regions on B7RP-1 to which 16H / 16Hg and 5D monoclonal antibodies bind. To this end, a novel fluorescence-activated cell sorter (FACS) binding assay was developed. Human extracellular domain (ECD) of B7RP1 (SEQ ID NO: 66) as well as B7RP-1 comprising Ig1 (IgV-like; SEQ ID NO: 67) or Ig2 (IgC-like; SEQ ID NO: 68) The truncated form was expressed as N-terminus in in-frame fusion with chicken avidin.

Expression vectors containing the gene encoding the fusion protein were temporarily transfected individually into 293T cells (ATCC #CRL 1573) and conditioned media from the cell line was used as a source of fusion protein. Avidin-tags were used to capture B7RP1 fusion proteins from solution using biotin-coated beads. Fusion proteins were incubated with fluorescently-labeled 16H or 5D mAbs or fluorescently-labeled ICOS-Fc fusion proteins and incubated with biotin-coated beads. Beads were recovered and analyzed using flow cytometry on a Becton-Dickinson Bioscience FACScan (BD, Franklin Lakes, NJ). As shown in FIG . 9A , the fluorescence staining of beads when the complete ECD of B7RP-1 was bound was detected using 16H, 5D and ICOS reagents, all three of which could bind to the ECD of B7RP-1 Suggests. Similarly, all three reagents bound to an avidin fusion protein containing only the Ig1 domain, suggesting that ICOS and blocking anti-B7RP-1 mAbs could bind to this region. In contrast, neither ICOS nor anti-B7RP-1 mAbs could bind to a fusion protein comprising only the membrane-proximity Ig2 domain. Thus, the ICOS, 16H and 5D binding regions on B7RP-1 were located in the Ig1 domain.

Antibodies generated as described above in Example 1 and tested using an avidin fusion binding assay can be divided into two classes, epitope classes, H and D, as shown in Table 5. Of the 100 antibodies initially screened based on their ability to bind B7RP1, 15 failed to bind in the avidin fusion binding assay, most of which was due to degradation.

Example  9

SNP  Identification and Functional Analysis

Major single nucleotide polymorphism (SNP) variants were identified in B7RP-1 present in the population with an allele frequency of 28.4% ( FIG. 7 ). Variants were identified in mature protein coding sequences. A search of the National Center for Biotechnology Information (NCBI) databank revealed a second SNP variant; The second variant was identified in 1.5 individual (three chromosome) assays. The first SNP variant (V128I) was located in the first IgV-like domain and the NCBI SNP variant (L221F) was located in the second IgC-like domain.

As discussed above, both 16H and 5D monoclonal antibodies bind to the first IgV-like domain, and the latter L221F variant does not seem to affect 16H or 5D mAb binding or function. Nevertheless, two different experiments were conducted to determine whether these SNP variants affected 16H or 5D binding and / or function. In the experiment of the first set up, avidin fusion proteins were constructed using two SNP variants and tested for binding to 16H or 5D antibodies in a flow cytometry assay as described above. Representative mAbs from H and D epitope classes that bind to SNP variants have similar efficacy as wild type B7RP-1 ( FIG. 9B ). The data suggest that antibodies from both H and D epitope classes bind to B7RP-1 SNP variants.

In a second approach, Fc fusion proteins were constructed using B7RP-1 SNP variant sequences and compared for their ability to stimulate T cells in a plate costimulatory assay ( FIG. 9C ). Both 16H and 5D antibodies inhibit co-stimulation mediated by SNP variant Fc fusion proteins with EC ₅₀ similar to wild-type fusion proteins. Aggregating the data suggests that two potential B7RP-1 SNP variants are recognized by the antibodies of the invention. Accordingly, the antibody of the present invention can bind to a target in a patient comprising the SNP variant.

Example  10

In vivo   Animal efficacy model

The ability of B7RP-1 antibodies to inhibit the immune response was achieved using the murine ratized anti-murine B7RP-1 monoclonal antibody (1B7v2) and keyhole-limpet hemocyanin (KLH). It was analyzed by challenge with BALB / c mice.

Rat   Rat Rat   B7RP-1 Monoclonal   Generation of Antibody 1B7v2

Chinese hamster ovary cells overexpressing full-length murine B7RP-1 were injected as primary immunizations in rats and subsequently the murine B7RP-1-Fc fusion protein was used to increase the immune response. Spleens were harvested on day 3 or 4 after intravenous booster immunization and splenic B cells were fused with Y3-Ag1.2.3 rat myeloma cell line (ATCC CRL-1631). Cells were then selected for 2 weeks in medium supplemented with hypoxanthine-aminopterin-thymidine (HAT), followed by subcloning of single cells to dilution limit. The procedures are described in "Practical Immunology, 2nd ed." Leslie Hudson and Frank C. Hay; Blackwell Scientific Publications 1980.

The gene encoding the 1B7 immunoglobulin was coded for the 1B7 cell line (hB7RP-1) using standard procedures (Sambrook et al. 2001, MOLECULAR CLONING: A LABORATORY MANUAL, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). To a CHO cell line transfected with DNA). The isotype switch of human anti-huB7RP1 MAbs consists of a variable region fragment comprising Xba I and Bsm BI restriction site binding ends and a pDSRα20 vector with human IgG1 or huIgG2 constant regions also having Xba I and Bsm BI ends. Completed by cloning. For 1B7 rat anti-muB7RP1, chimeras were formed by a three step overlap PCR process. Rat variable regions were PCR amplified using 3 ′ primers comprising a portion of about 25 to 35 nucleotides of the murine constant region. The murine constant region was PCR amplified using a 5 'primer comprising a portion of about 25 to 35 nucleotides of the rat variable region. Two fragments were then used as templates and 5 'rat variable region (including Xba I) and murine 3' constant region (including Sal I) primers were used for complete light or heavy chain generation. The light and heavy chain PCR products were then cleaved with Xba I and Sal I and cloned into pDSRa19. A total of 25 μg of linearized DNA (12.5 μg pDC323B LC + 12.5 μg pDC324 HC) was transfected into CS-9 cells using electrophoresis and selected in DHFR-supplement medium. The CS-9 cells were derived from CHO DUX B11 (obtained from Lawrence Chasin, Columbia University) through standard techniques of growing and sub-cloning in serum-free medium.

To test the efficacy of the 1B7v2 mAb, a plate costimulatory assay was performed using the mAb. Results were compared to other anti-murine B7RP-1 mAb (FIG. 10A). As discussed above, 1B7 is the original hybridoma-producing mAb; Two different formulations (labeled 1.33 and 7.4) were tested. 5E1 and 11G10 are other anti-mB7RP-1 monoclonals produced in the fusions described above. Finally, HK5.3 is a commercially available anti-mB7RP-1 (ebiosciences # 16-5985-85).

1B7v2 mAb blocks T cell activation in this assay equal to or greater than any other mAb and thus was selected as a surrogate treatment for further studies.

Antigen Administration in Mice

Keyhole Limpet Hemocyanin (KLH) is sold by Pierce Biotechnology (Rockford, Illinois). Dosing solution # 1 (1 mg / mouse 5 mg / kg of mouse ALUM) contained the same portion of 2x ALUM (500 mg ALUM and 50 ml PBS (phosphate buffered saline)) and 2x KLH (2.0 ml dH20 (RNAse-Free)). 20 mg of lyophilized KLH and then 20 mL of 1 × PBS were prepared). Dosing solution # 2 (1 mg / kg KLH in 1 mg / mouse ALUM) was prepared with 1 part 2 × KLH mixed with 4 parts 1 × phosphate buffered saline.

Female BALB / c mice were pretreated with 1 mg / kg KLH / alum and reimmunized on day 21 by intraperitoneal injection with only 5 mg / kg KLH. Mice were intraperitoneally injected every 5 days starting at Day 1 (one day before pretreatment with KLH / alum) at 200 μl final volume with 1B7v.2, isotype control antibody (anti-AGP3 PB) or vehicle (PBS) only did.

Retro-orbitally every 7 days in mice to obtain about 50-100 μl of serum for analysis of antigen-specific serum IgM ( FIG. 10B ), IgG2a ( FIG. 10C ) and IgG1 ( FIG. 10D ). Blood was collected (approximately 200 μl). Both isotype-control and vehicle treated mice showed significant primary and secondary immune responses. IgM response is not affected by treatment, but blocking of B7RP-1-ICOS with 1B7v2 reduces both primary and secondary IgG2a and IgG1 responses in a statistically significant manner.

IL-5 is a cytokine released by T cells in response to antigenic stimulation that induces B cell differentiation and function. Since B7RP-1 / ICOS interactions are believed to be important for T-cell-dependent B cell function, serum IL-5 level measurements can determine whether inhibition of the B7RP-1 / ICOS axis actually affects T cell function. Used for. As expected, blocking of B7RP-1 also inhibited antigen induced serum IL-5 levels. Serum was summed over 24 hours on day 21 of challenge from mice in the challenge experiments outlined serum and serum IL-5 levels were determined by ELISA. As shown in FIG . 11 , elevated IL-5 levels were detected in test mice as soon as 9 hours after challenge; Levels began to decline by 48 hours and returned to baseline by 72 hours. Treatment of mice with 1B7v2 mAb leads to a statistically significant decrease in IL-5 levels at 24-hour time points.

Example  11

Cynomolgus   Binding to monkey B7RP-1

To determine whether anti-hB7RP-1 mAbs also bind to cynomolgus monkey B7RP-1, flow cytometry comprising experiments was performed using 16H mAb and B cells purified from cynomolgus monkeys and humans. . As shown in FIG . 12A , the addition of fluorescently labeled 16H to cyno B cells induces staining, suggesting that 16H is indeed bound to cyno B7RP-1 (right panel). As expected, 16H also stained human B cells (left panel). In addition, 16H, 16Hg and 5D were tested in plate costimulatory assays using cyno T cells, cyno B7RP-1-Fc and anti-CD3 mAb. As shown in FIG . 12B , all three mAbs inhibited cyno B7RP-1-dependent cyno T cell activation, suggesting that mAb functionally blocks cyno ICOS-B7RP-1 interaction.

Example  12

After administration of anti-B7RP-1 antibody Cynomolgus Monkey   T-cell dependent antigen response

Cynomolgus monkey studies were performed using two anti-B7RP-1 monoclonal antibodies, 16H and 5D, whereby T-cell dependent B cells as determined by the serum levels of antigen-specific antibodies The ability to inhibit antigen response was evaluated. Briefly, anti-kipol limpet hemocyanin (KLH) and anti-tetanus toxoid antibody responses were tested after challenge in the presence of B7RP-1 antibodies in cynomolgus monkeys.

Test substance 1 was 16H and test substance 2 was 5D. The control object was the vehicle for B7RP-1 antibody (0.01 sodium acetate, pH 5.0, 5% sorbitol, 0.004% Tween 20). Keyhole Limpet Hemocyanin (KLH) is sold by Pierce Biotechnology (Rockford, Illinois).

KLH was prepared by reconstitution with sterile water to provide a 10 mg / mL stock solution. The stock solution was diluted with sterile water to provide a 1 mg / mL dose solution. Tetanus toxoid used in the experiment is Intervet ^TM Inc. Super-Tet ^® Tetanous Toxoid w / Havlogen ^® sold by Milsboro, Delaware. The dose level for this experiment was 75 IU (0.5 mL of 150 IU / mL).

Table 6 shows the treatment group distribution of 28 cynomolgus monkeys.

Test substance doses were administered via intravenous injection to all animals on days 1, 8, 15, 22, 29, 36, 43 and 50. Animals scheduled for necropsy (groups / gender / group) in groups 1-4 received additional dose on day 57. Evaluation of the immune response was performed in all animals via immunization with KLH and tetanus toxoid antigens, followed by blood sampling for antigen-specific immunoglobulins (IgM and IgG).

Titers were present after primary administration of both KLH and tetanus antigens. For KLH, the primary titers ranged from 0 to 900 for both IgM and IgG. Primary KLH antigens were administered prior to test substance administration and were assessed to have no effect of the B7RP-1 antibody. For tetanus toxoid, the primary titers range from 0 to 50 for IgM and from 0 to 4050 for IgG. There was no difference in the primary response to tetanus toxoid between the B7RP-1 antibody groups and the control group.

As expected, titers to IgG increased compared to primary titers after secondary administration of both KLH and tetanus antigens. For KLH, the secondary titers ranged from 0 to 300 for IgM and from 0 to 8100 for IgG. However, there was no evidence of inhibition of the KLH secondary response resulting in the administration of the B7RP-1 antibody.

For tetanus toxoid, the secondary titers were less than 50 for IgM and 1350 to 36450 for IgG. The results of individual animal and group mean values are shown in FIGS. 13A (16H antibody) and 13B (5D antibody) for days 53 and 57 after secondary challenge with tetanus toxoid on day 42.

On day 53, the population of animals reaching the peak response was 3/4, 1/4 and 1 for 16H at 0.1, 1 and 8 mg / kg dose levels, respectively, compared to 2/4 in control animals. / 4 and 1/4, 1/4 and 1/4 respectively for 0.1, 1 and 8 mg / kg dose levels of 5D. Thus, in general, the population of animals that reached high titers on day 53 decreased in the B7RP-1 antibody-treated group. On day 57, titers were maintained in control animals and in some of the B7RP-1 antibody-treated animals the titers decreased below the day 53 values. The number of animals with high titers on day 57 was 0/4, 0/4 and 1/4 respectively at 16H at 0.1, 1 and 8 mg / kg dose levels compared to 2/4 in control animals. And at 4D at 0.1, 1 and 8 mg / kg dose levels, 0/4, 0/4, and 0/4, respectively.

The results demonstrated that two B7RP-1 antibodies 16H and 5D inhibit T-cell dependent B cell antigen responses measured by serum levels of tetanus toxoid-specific antibodies in cynomolgus monkeys. In addition, the presence of B7RP-1 antibodies is important for blocking of B7RP-1-ICOS interactions during the primary reaction to detect the effects after secondary challenge.

The results and results of Example 10 demonstrate that both the therapeutic and therapeutic candidates block T and B cell dependent immune responses in murine and monkey model systems, such that blocking the costimulatory axis is a B-cell mediated disease, such as It may suggest that it may be effective in the treatment of systemic lupus erythematosus (SLE), asthma and rheumatoid arthritis (RA).

It is to be understood that the above disclosure highlights specific specific embodiments of the invention, and all modifications or alternative equivalents thereof are included in the spirit and scope of the invention as set forth in the appended claims.

SEQUENCE LISTING <110> Siu, Jerry        Shen, David        Yoshinaga, Steve        Huang, Haichun   <120> Human anti-B7RP1 Neutralizing Antibodies <130> 04-833 <160> 76 <170> PatentIn version 3.3 <210> 1 <211> 107 <212> PRT <213> Homo sapiens <400> 1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Trp             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile         35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ser Tyr Pro Arg                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 2 <211> 107 <212> PRT <213> Homo sapiens <400> 2 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile         35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Asn Asn Trp Pro Trp                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 3 <211> 107 <212> PRT <213> Homo sapiens <400> 3 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Trp             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile         35 40 45 Tyr Thr Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Arg                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 4 <211> 108 <212> PRT <213> Homo sapiens <400> 4 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile         35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ala                 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 5 <211> 107 <212> PRT <213> Homo sapiens <400> 5 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile         35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Thr Tyr Pro Arg                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 6 <211> 107 <212> PRT <213> Homo sapiens <400> 6 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile         35 40 45 Phe Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys             100 105   <210> 7 <211> 121 <212> PRT <213> Homo sapiens <400> 7 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Tyr Ile Lys Gln Asp Gly Asn Glu Lys Tyr Tyr Val Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Glu Gly Ile Leu Trp Phe Gly Asp Leu Pro Thr Phe Trp Gly             100 105 110 Gln Gly Ile Leu Val Thr Val Ser Ser         115 120 <210> 8 <211> 121 <212> PRT <213> Homo sapiens <400> 8 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Tyr Ile Lys Gln Asp Gly Asn Glu Lys Tyr Tyr Val Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Glu Gly Ile Leu Trp Phe Gly Asp Leu Pro Thr Phe Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 9 <211> 121 <212> PRT <213> Homo sapiens <400> 9 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Ala Asp Ser Val     50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Phe Tyr Cys                 85 90 95 Ala Arg Asp Leu Asn Ile Met Val Trp Gly Ile Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 10 <211> 121 <212> PRT <213> Homo sapiens <400> 10 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Gly Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Tyr Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Asp Gly Ile Leu Trp Phe Gly Asp Ile Pro Thr Tyr Trp Gly             100 105 110 Gln Gly Ile Leu Val Thr Val Ser Ser         115 120 <210> 11 <211> 121 <212> PRT <213> Homo sapiens <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Tyr Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Asp Gly Ile Leu Trp Phe Gly Asp Ile Pro Thr Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 12 <211> 120 <212> PRT <213> Homo sapiens <400> 12 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val His Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Met Tyr Tyr Cys Ala                 85 90 95 Lys Ser Gly Val Pro Leu Leu Trp Phe Gly Glu Phe Tyr Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 13 <211> 121 <212> PRT <213> Homo sapiens <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Tyr Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Asp Gly Ile Leu Trp Phe Gly Asp Leu Pro Thr Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 14 <211> 118 <212> PRT <213> Homo sapiens <400> 14 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val His Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Ala Ile Gly Ala Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Val Val Val Val Val Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser         115 <210> 15 <211> 11 <212> PRT <213> Homo sapiens <400> 15 Arg Ala Ser Gln Gly Ile Ser Asn Trp Leu Ala 1 5 10 <210> 16 <211> 7 <212> PRT <213> Homo sapiens <400> 16 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 17 <211> 9 <212> PRT <213> Homo sapiens <400> 17 Gln Gln Tyr Asp Ser Tyr Pro Arg Thr 1 5 <210> 18 <211> 11 <212> PRT <213> Homo sapiens <400> 18 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 19 <211> 7 <212> PRT <213> Homo sapiens <400> 19 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 20 <211> 9 <212> PRT <213> Homo sapiens <400> 20 Gln Gln Arg Asn Asn Trp Pro Trp Thr 1 5 <210> 21 <211> 7 <212> PRT <213> Homo sapiens <400> 21 Thr Ala Ser Ser Leu Gln Ser 1 5 <210> 22 <211> 9 <212> PRT <213> Homo sapiens <400> 22 Gln Gln Tyr Asn Ser Tyr Pro Arg Thr 1 5 <210> 23 <211> 10 <212> PRT <213> Homo sapiens <400> 23 Gln Gln Arg Ser Asn Trp Pro Ala Leu Thr 1 5 10 <210> 24 <211> 11 <212> PRT <213> Homo sapiens <400> 24 Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala 1 5 10 <210> 25 <211> 9 <212> PRT <213> Homo sapiens <400> 25 Gln Gln Tyr Asn Thr Tyr Pro Arg Thr 1 5 <210> 26 <211> 9 <212> PRT <213> Homo sapiens <400> 26 Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr 1 5 <210> 27 <211> 5 <212> PRT <213> Homo sapiens <400> 27 Ser Tyr Trp Met Ser 1 5 <210> 28 <211> 17 <212> PRT <213> Homo sapiens <400> 28 Tyr Ile Lys Gln Asp Gly Asn Glu Lys Tyr Tyr Val Asp Ser Val Lys 1 5 10 15 Gly      <210> 29 <211> 12 <212> PRT <213> Homo sapiens <400> 29 Glu Gly Ile Leu Trp Phe Gly Asp Leu Pro Thr Phe 1 5 10 <210> 30 <211> 5 <212> PRT <213> Homo sapiens <400> 30 Ser Tyr Gly Met His 1 5 <210> 31 <211> 17 <212> PRT <213> Homo sapiens <400> 31 Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Ala Asp Ser Val Glu 1 5 10 15 Gly      <210> 32 <211> 12 <212> PRT <213> Homo sapiens <400> 32 Asp Leu Asn Ile Met Val Trp Gly Ile Phe Asp Tyr 1 5 10 <210> 33 <211> 17 <212> PRT <213> Homo sapiens <400> 33 Tyr Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Lys 1 5 10 15 Gly      <210> 34 <211> 12 <212> PRT <213> Homo sapiens <400> 34 Asp Gly Ile Leu Trp Phe Gly Asp Ile Pro Thr Tyr 1 5 10 <210> 35 <211> 5 <212> PRT <213> Homo sapiens <400> 35 Ser Tyr Ala Met His 1 5 <210> 36 <211> 16 <212> PRT <213> Homo sapiens <400> 36 Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 37 <211> 11 <212> PRT <213> Homo sapiens <400> 37 Gly Val Pro Leu Leu Trp Phe Gly Glu Phe Tyr 1 5 10 <210> 38 <211> 12 <212> PRT <213> Homo sapiens <400> 38 Asp Gly Ile Leu Trp Phe Gly Asp Leu Pro Thr Tyr 1 5 10 <210> 39 <211> 16 <212> PRT <213> Homo sapiens <400> 39 Ala Ile Gly Ala Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 40 <211> 10 <212> PRT <213> Homo sapiens <400> 40 Val Val Val Val Val Gly Phe Phe Asp Tyr 1 5 10 <210> 41 <211> 326 <212> PRT <213> Homo sapiens <400> 41 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr             20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser         35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser     50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys                 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro             100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp         115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp     130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn                 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp             180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro         195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu     210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile                 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr             260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys         275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys     290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys                 325 <210> 42 <211> 330 <212> PRT <213> Homo sapiens <400> 42 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr             20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser         35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser     50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys                 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys             100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro         115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys     130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu                 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu             180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn         195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly     210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr                 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn             260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe         275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn     290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys                 325 330 <210> 43 <211> 107 <212> PRT <213> Homo sapiens <400> 43 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe             20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln         35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser     50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser                 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys             100 105 <210> 44 <211> 447 <212> PRT <213> Homo sapiens <400> 44 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Tyr Ile Lys Gln Asp Gly Asn Glu Lys Tyr Tyr Val Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Glu Gly Ile Leu Trp Phe Gly Asp Leu Pro Thr Phe Trp Gly             100 105 110 Gln Gly Ile Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser         115 120 125 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala     130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala                 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val             180 185 190 Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His         195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys     210 215 220 Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr                 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu             260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys         275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser     290 295 300 Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile                 325 330 335 Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro             340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu         355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn     370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg                 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu             420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         435 440 445 <210> 45 <211> 214 <212> PRT <213> Homo sapiens <400> 45 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Trp             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile         35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ser Tyr Pro Arg                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 46 <211> 447 <212> PRT <213> Homo sapiens <400> 46 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Tyr Ile Lys Gln Asp Gly Asn Glu Lys Tyr Tyr Val Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Glu Gly Ile Leu Trp Phe Gly Asp Leu Pro Thr Phe Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser         115 120 125 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala     130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala                 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val             180 185 190 Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His         195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys     210 215 220 Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr                 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu             260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys         275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser     290 295 300 Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile                 325 330 335 Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro             340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu         355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn     370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg                 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu             420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         435 440 445 <210> 47 <211> 451 <212> PRT <213> Homo sapiens <400> 47 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Ala Asp Ser Val     50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Phe Tyr Cys                 85 90 95 Ala Arg Asp Leu Asn Ile Met Val Trp Gly Ile Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser         115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala     130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala                 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val             180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His         195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys     210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met                 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His             260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val         275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr     290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile                 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val             340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser         355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu     370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val                 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met             420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser         435 440 445 Pro Gly Lys     450 <210> 48 <211> 214 <212> PRT <213> Homo sapiens <400> 48 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile         35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Asn Asn Trp Pro Trp                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 49 <211> 433 <212> PRT <213> Homo sapiens <400> 49 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe             20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln         35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser     50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser                 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Ala Ser Thr Lys Gly             100 105 110 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser         115 120 125 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val     130 135 140 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 145 150 155 160 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val                 165 170 175 Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val             180 185 190 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys         195 200 205 Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro     210 215 220 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 225 230 235 240 Arg Thr Pro Glu Val Thr Cys Val Val Asp Val Ser His Glu Asp                 245 250 255 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn             260 265 270 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val         275 280 285 Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu     290 295 300 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys 305 310 315 320 Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr                 325 330 335 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr             340 345 350 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu         355 360 365 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu     370 375 380 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 385 390 395 400   Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu                 405 410 415 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly             420 425 430 Lys      <210> 50 <211> 214 <212> PRT <213> Homo sapiens <400> 50 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Trp             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile         35 40 45 Tyr Thr Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Arg                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 51 <211> 447 <212> PRT <213> Homo sapiens <400> 51 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Tyr Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95   Ala Arg Asp Gly Ile Leu Trp Phe Gly Asp Ile Pro Thr Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser         115 120 125 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala     130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala                 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val             180 185 190 Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His         195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys     210 215 220 Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr                 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu             260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys         275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser     290 295 300 Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile                 325 330 335 Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro             340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu         355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn     370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg                 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu             420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         435 440 445 <210> 52 <211> 444 <212> PRT <213> Homo sapiens <400> 52 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val His Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Ala Ile Gly Ala Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Val Val Val Val Val Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro         115 120 125 Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly     130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln                 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser             180 185 190 Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser         195 200 205 Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys     210 215 220 Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val                 245 250 255 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe             260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro         275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr     290 295 300 Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr                 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg             340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly         355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro     370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln                 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His             420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         435 440 <210> 53 <211> 214 <212> PRT <213> Homo sapiens <400> 53 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile         35 40 45 Phe Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 54 <211> 447 <212> PRT <213> Homo sapiens <400> 54 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Tyr Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Asp Gly Ile Leu Trp Phe Gly Asp Leu Pro Thr Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser         115 120 125 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala     130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala                 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val             180 185 190 Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His         195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys     210 215 220 Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr                 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu             260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys         275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser     290 295 300 Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile                 325 330 335 Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro             340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu         355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn     370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg                 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu             420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         435 440 445 <210> 55 <211> 214 <212> PRT <213> Homo sapiens <400> 55 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile         35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Thr Tyr Pro Arg                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 56 <211> 446 <212> PRT <213> Homo sapiens <400> 56 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val His Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Met Tyr Tyr Cys Ala                 85 90 95 Lys Ser Gly Val Pro Leu Leu Trp Phe Gly Glu Phe Tyr Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val         115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala     130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val                 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro             180 185 190 Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys         195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val     210 215 220 Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro                 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val             260 265 270 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr         275 280 285 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val     290 295 300 Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser                 325 330 335 Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro             340 345 350 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val         355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly     370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp                 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His             420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         435 440 445 <210> 57 <211> 450 <212> PRT <213> Homo sapiens <400> 57 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val His Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Met Tyr Tyr Cys Ala                 85 90 95 Lys Ser Gly Val Pro Leu Leu Trp Phe Gly Glu Phe Tyr Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val         115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala     130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val                 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro             180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys         195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp     210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile                 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu             260 265 270   Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His         275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg     290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu                 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr             340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu         355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp     370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp                 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His             420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro         435 440 445 Gly lys     450 <210> 58 <211> 215 <212> PRT <213> Homo sapiens <400> 58 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile         35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ala                 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala             100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser         115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu     130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu                 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val             180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys         195 200 205 Ser Phe Asn Arg Gly Glu Cys     210 215 <210> 59 <211> 24 <212> DNA <213> Homo sapiens <220> <221> misc_feature (222) (18) .. (23) N is a, c, g, or t <400> 59 ggccggatag gcctccannn nnnt 24 <210> 60 <211> 21 <212> DNA <213> Homo sapiens <400> 60 ggggtcaggc tggaactgag g 21 <210> 61 <211> 19 <212> DNA <213> Homo sapiens <400> 61 tgaggacgct gaccacacg 19 <210> 62 <211> 54 <212> DNA <213> Homo sapiens <400> 62 cagcagaagc ttctagacca ccatggacat gagggtcctc gctcagctcc tggg 54 <210> 63 <211> 34 <212> DNA <213> Homo sapiens <400> 63 cttgtcgact caacactctc ccctgttgaa gctc 34 <210> 64 <211> 43 <212> DNA <213> Homo sapiens <400> 64 acaacaaagc ttctagacca ccatggagtt ggggctgaac tgg 43 <210> 65 <211> 32 <212> DNA <213> Homo sapiens <400> 65 gtggaggcac tagagacggt gaccaggatt cc 32 <210> 66 <211> 228 <212> PRT <213> Homo sapiens <400> 66 Asp Thr Gln Glu Lys Glu Val Arg Ala Met Val Gly Ser Asp Val Glu 1 5 10 15 Leu Ser Cys Ala Cys Pro Glu Gly Ser Arg Phe Asp Leu Asn Asp Val             20 25 30 Tyr Val Tyr Trp Gln Thr Ser Glu Ser Lys Thr Val Val Thr Tyr His         35 40 45 Ile Pro Gln Asn Ser Ser Leu Glu Asn Val Asp Ser Arg Tyr Arg Asn     50 55 60 Arg Ala Leu Met Ser Pro Ala Gly Met Leu Arg Gly Asp Phe Ser Leu 65 70 75 80 Arg Leu Phe Asn Val Thr Pro Gln Asp Glu Gln Lys Phe His Cys Leu                 85 90 95 Val Leu Ser Gln Ser Leu Gly Phe Gln Glu Val Leu Ser Val Glu Val             100 105 110 Thr Leu His Val Ala Ala Asn Phe Ser Val Pro Val Val Ser Ala Pro         115 120 125 His Ser Pro Ser Gln Asp Glu Leu Thr Phe Thr Cys Thr Ser Ile Asn     130 135 140 Gly Tyr Pro Arg Pro Asn Val Tyr Trp Ile Asn Lys Thr Asp Asn Ser 145 150 155 160 Leu Leu Asp Gln Ala Leu Gln Asn Asp Thr Val Phe Leu Asn Met Arg                 165 170 175 Gly Leu Tyr Asp Val Val Ser Val Leu Arg Ile Ala Arg Thr Pro Ser             180 185 190 Val Asn Ile Gly Cys Cys Ile Glu Asn Val Leu Leu Gln Gln Asn Leu         195 200 205 Thr Val Gly Ser Gln Thr Gly Asn Asp Ile Gly Glu Arg Asp Lys Ile     210 215 220 Thr Glu Asn Pro 225 <210> 67 <211> 139 <212> PRT <213> Homo sapiens <400> 67 Asp Thr Gln Glu Lys Glu Val Arg Ala Met Val Gly Ser Asp Val Glu 1 5 10 15 Leu Ser Cys Ala Cys Pro Glu Gly Ser Arg Phe Asp Leu Asn Asp Val             20 25 30 Tyr Val Tyr Trp Gln Thr Ser Glu Ser Lys Thr Val Val Thr Tyr His         35 40 45 Ile Pro Gln Asn Ser Ser Leu Glu Asn Val Asp Ser Arg Tyr Arg Asn     50 55 60 Arg Ala Leu Met Ser Pro Ala Gly Met Leu Arg Gly Asp Phe Ser Leu 65 70 75 80 Arg Leu Phe Asn Val Thr Pro Gln Asp Glu Gln Lys Phe His Cys Leu                 85 90 95 Val Leu Ser Gln Ser Leu Gly Phe Gln Glu Val Leu Ser Val Glu Val             100 105 110 Thr Leu His Val Ala Ala Asn Phe Ser Val Pro Val Val Ser Ala Pro         115 120 125 His Ser Pro Ser Gln Asp Glu Leu Thr Phe Thr     130 135 <210> 68 <211> 127 <212> PRT <213> Homo sapiens <400> 68 Leu Gly Phe Gln Glu Val Leu Ser Val Glu Val Thr Leu His Val Ala 1 5 10 15 Ala Asn Phe Ser Val Pro Val Val Ser Ala Pro His Ser Pro Ser Gln             20 25 30 Asp Glu Leu Thr Phe Thr Cys Thr Ser Ile Asn Gly Tyr Pro Arg Pro         35 40 45 Asn Val Tyr Trp Ile Asn Lys Thr Asp Asn Ser Leu Leu Asp Gln Ala     50 55 60 Leu Gln Asn Asp Thr Val Phe Leu Asn Met Arg Gly Leu Tyr Asp Val 65 70 75 80 Val Ser Val Leu Arg Ile Ala Arg Thr Pro Ser Val Asn Ile Gly Cys                 85 90 95 Cys Ile Glu Asn Val Leu Leu Gln Gln Asn Leu Thr Val Gly Ser Gln             100 105 110 Thr Gly Asn Asp Ile Gly Glu Arg Asp Lys Ile Thr Glu Asn Pro         115 120 125 <210> 69 <211> 39 <212> DNA <213> Homo sapiens <400> 69 cgacggagca cgaggacacg acaggacgaa ggagagaaa 39 <210> 70 <211> 451 <212> PRT <213> Homo sapiens <400> 70 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Tyr Ile Lys Gln Asp Gly Asn Glu Lys Tyr Tyr Val Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Glu Gly Ile Leu Trp Phe Gly Asp Leu Pro Thr Phe Trp Gly             100 105 110 Gln Gly Ile Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser         115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala     130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala                 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val             180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His         195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys     210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met                 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His             260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val         275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr     290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile                 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val             340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser         355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu     370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val                 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met             420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser         435 440 445 Pro Gly Lys     450 <210> 71 <211> 451 <212> PRT <213> Homo sapiens <400> 71 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Tyr Ile Lys Gln Asp Gly Asn Glu Lys Tyr Tyr Val Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Glu Gly Ile Leu Trp Phe Gly Asp Leu Pro Thr Phe Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser         115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala     130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala                 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val             180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His         195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys     210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met                 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His             260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val         275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr     290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile                 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val             340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser         355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu     370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val                 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met             420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser         435 440 445 Pro Gly Lys     450 <210> 72 <211> 447 <212> PRT <213> Homo sapiens <400> 72 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Lys Lys Tyr Tyr Ala Asp Ser Val     50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Phe Tyr Cys                 85 90 95 Ala Arg Asp Leu Asn Ile Met Val Trp Gly Ile Phe Asp Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser         115 120 125 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala     130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala                 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val             180 185 190 Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His         195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys     210 215 220 Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr                 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu             260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys         275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser     290 295 300 Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile                 325 330 335 Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro             340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu         355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn     370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg                 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu             420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         435 440 445 <210> 73 <211> 451 <212> PRT <213> Homo sapiens <400> 73 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15   Ser Leu Arg Leu Ser Cys Val Gly Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Tyr Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Asp Gly Ile Leu Trp Phe Gly Asp Ile Pro Thr Tyr Trp Gly             100 105 110 Gln Gly Ile Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser         115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala     130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala                 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val             180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His         195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys     210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met                 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His             260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val         275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr     290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile                 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val             340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser         355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu     370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val                 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met             420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser         435 440 445 Pro Gly Lys     450 <210> 74 <211> 451 <212> PRT <213> Homo sapiens <400> 74 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Tyr Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Asp Gly Ile Leu Trp Phe Gly Asp Ile Pro Thr Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser         115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala     130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala                 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val             180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His         195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys     210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met                 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His             260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val         275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr     290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile                 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val             340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser         355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu     370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val                 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met             420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser         435 440 445 Pro Gly Lys     450 <210> 75 <211> 448 <212> PRT <213> Homo sapiens <400> 75 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val His Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Ala Ile Gly Ala Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys     50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Val Val Val Val Val Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro         115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly     130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln                 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser             180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser         195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr     210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg                 245 250 255 Thr Pro Glu Val Thr Cys Val Val Asp Val Ser His Glu Asp Pro             260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala         275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val     290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr                 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu             340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys         355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser     370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser                 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala             420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         435 440 445 <210> 76 <211> 451 <212> PRT <213> Homo sapiens <400> 76 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Tyr Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Asp Gly Ile Leu Trp Phe Gly Asp Leu Pro Thr Tyr Trp Gly             100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser         115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala     130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala                 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val             180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His         195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys     210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met                 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His             260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val         275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr     290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile                 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val             340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser         355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu     370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val                 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met             420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser         435 440 445 Pro Gly Lys     450  

Claims (87)

An isolated antibody that specifically binds to B7RP1, including heavy and light chains with heavy chain variable regions, wherein the heavy chain variable region has at least 80% sequence identity with the amino acid sequence set forth in any of SEQ ID NOs: 7-14 An isolated antibody comprising an amino acid sequence having, or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment. The isolated antibody of claim 1, wherein the antibody specifically binds to human B7RP1 but not to mouse B7RP1. The isolated antibody of claim 1, wherein the antibody specifically binds human B7RP1 and mouse B7RP1. The antibody of claim 1, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NOs: 7-14, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment. The antibody of claim 1, wherein the heavy chain comprises a human heavy chain CDR1 having an amino acid sequence as set forth in SEQ ID NO: 27, 30, or 35, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment. . The heavy chain of claim 1, wherein the heavy chain comprises a human heavy chain CDR3 having an amino acid sequence set forth in SEQ ID NO: 29, 32, 34, 37, 38, or 40, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment. Characterized in that the antibody. The method of claim 1, wherein the heavy chain comprises a human heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 28, 31, 33, 36 or 39, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment. Characterized in the antibody. An isolated antibody that specifically binds B7RP1, including a) a heavy chain having a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 7, or an antigen binding fragment or immunologically functional immunoglobulin fragment, and an amino acid sequence set forth in SEQ ID NO: 1, or A light chain having a light chain variable region comprising an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment; b) a heavy chain having a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 8, or an antigen binding fragment or immunologically functional immunoglobulin fragment, and an amino acid sequence set forth in SEQ ID NO: 1, or A light chain having a light chain variable region comprising an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment; c) a heavy chain having a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 9, or an antigen binding fragment or immunologically functional immunoglobulin fragment, and an amino acid sequence set forth in SEQ ID NO: 2, or A light chain having a light chain variable region comprising an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment; d) a heavy chain having a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 10, or an antigen binding fragment or immunologically functional immunoglobulin fragment, and an amino acid sequence set forth in SEQ ID NO: 3, or A light chain having a light chain variable region comprising an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment; e) a heavy chain having a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 11, or an antigen binding fragment or immunologically functional immunoglobulin fragment, and an amino acid sequence set forth in SEQ ID NO: 3, or A light chain having a light chain variable region comprising an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment; f) a heavy chain having a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 12, or an antigen binding fragment or immunologically functional immunoglobulin fragment, and an amino acid sequence set forth in SEQ ID NO: 4, or A light chain having a light chain variable region comprising an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment; g) a heavy chain having a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 13, or an antigen binding fragment or immunologically functional immunoglobulin fragment, and an amino acid sequence set forth in SEQ ID NO: 5, or A light chain having a light chain variable region comprising an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment; or h) a heavy chain having a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 14, or an antigen binding fragment or immunologically functional immunoglobulin fragment, and an amino acid sequence set forth in SEQ ID NO: 6, or A light chain having a light chain variable region comprising an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment. An isolated antibody that specifically binds B7RP1, wherein the antibody comprises a light and heavy chain having a light chain variable region, wherein the light chain variable region is at least 80% of the amino acid sequence set forth in SEQ ID NOs: 1-6 An isolated antibody comprising an amino acid sequence having identity, or an antigen binding fragment or immunogenic fragment thereof. The isolated antibody of claim 9, wherein the antibody specifically binds to human B7RP1 but not to mouse B7RP1. The isolated antibody of claim 9, wherein the antibody specifically binds human B7RP1 and mouse B7RP1. The light chain variable region of claim 9, wherein the light chain variable region comprises an amino acid sequence having at least 90% sequence identity with an amino acid sequence set forth in SEQ ID NOs: 1-6, or an antigen binding fragment or immunologically functional immunoglobulin fragment thereof Characterized in that the antibody. The antibody of claim 9, wherein the light chain comprises a human light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 15, 18, or 24, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment. . The human light chain CDR3 of claim 9, wherein the light chain comprises an amino acid sequence set forth in SEQ ID NO: 17, 20, 22, 23, 25 or 26, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment. Antibody, characterized in that. 10. The antibody of claim 9, wherein the light chain comprises a human light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 16, 19 or 21, or an antigen binding fragment or immunologically functional immunoglobulin fragment thereof. . An isolated antibody that specifically binds B7RP1, including a) heavy chain CDR1 has the amino acid sequence shown in SEQ ID NO: 27, heavy chain CDR2 has the amino acid sequence shown in SEQ ID NO: 28, and heavy chain CDR 3 has the amino acid sequence shown in SEQ ID NO: 29 ; b) heavy chain CDR1 has the amino acid sequence shown in SEQ ID NO: 30, heavy chain CDR2 has the amino acid sequence shown in SEQ ID NO: 31, and heavy chain CDR 3 has the amino acid sequence shown in SEQ ID NO: 32 ; c) heavy chain CDR1 has the amino acid sequence shown in SEQ ID NO: 27, heavy chain CDR2 has the amino acid sequence shown in SEQ ID NO: 33, and heavy chain CDR 3 has the amino acid sequence shown in SEQ ID NO: 34 ; d) heavy chain CDR1 has the amino acid sequence shown in SEQ ID NO: 35, heavy chain CDR2 has the amino acid sequence shown in SEQ ID NO: 36, and heavy chain CDR 3 has the amino acid sequence shown in SEQ ID NO: 37 ; e) heavy chain CDR1 has the amino acid sequence shown in SEQ ID NO: 27, heavy chain CDR2 has the amino acid sequence shown in SEQ ID NO: 33, heavy chain CDR 3 has the amino acid sequence shown in SEQ ID NO: 38 ; or f) heavy chain CDR1 has the amino acid sequence shown in SEQ ID NO: 35, heavy chain CDR2 has the amino acid sequence shown in SEQ ID NO: 39, and heavy chain CDR 3 has the amino acid sequence shown in SEQ ID NO: 40. . 17. The antibody of any one of claims 1, 9 and 16, wherein the heavy and light chains are joined by a flexible linker to form a single chain antibody. 18. The antibody of claim 17 which is a single chain Fv antibody. 17. The antibody of any one of claims 1, 9 and 16, which is a Fab antibody. The antibody according to any one of claims 1, 9 and 16, characterized in that it is a Fab 'antibody. The antibody according to any one of claims 1, 9 and 16, which is a (Fab ') ₂ antibody. The antibody of any one of claims 1, 9 and 16, wherein the antibody is a fully human antibody. 17. The antibody of any one of claims 1, 9 and 16, wherein the antibody inhibits B7RP1 activity. A method of treating an autoimmune disease or an inflammatory response by administering to a patient a pharmaceutically effective amount of an isolated antibody that specifically binds B7RP1, wherein the antibody comprises any of SEQ ID NOs: 1-40, 44-58 or 70 A method of treating an autoimmune disease or an inflammatory response comprising the amino acid sequence set forth at -76, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment. The method of claim 24, wherein the autoimmune disease is rheumatoid arthritis or systemic lupus erythematosus. The method of claim 24, wherein the inflammatory response is asthma. A method of inhibiting costimulatory signal T-cell activation in a patient by administering to the patient a pharmaceutically effective amount of an isolated human antibody that specifically binds to B7RP1, wherein the antibody comprises any of SEQ ID NOs: 1-40, A method comprising the amino acid sequence set forth in 44-58, or 70-76, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment. A method of treating a patient suffering from asthma, rheumatoid arthritis or systemic lupus erythematosus by administering to a patient a pharmaceutically effective amount of an isolated human antibody that specifically binds B7RP1, wherein the antibody is any of SEQ ID NO: 1 A method comprising the amino acid sequence set forth at -40, 44-58 or 70-76, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment. A pharmaceutical composition comprising a pharmaceutically effective amount of an isolated antibody that binds specifically to B7RP1 and a pharmaceutically acceptable carrier, wherein the antibody comprises any of SEQ ID NOs: 1-40, 44-58 or 70-76. A pharmaceutical composition comprising an amino acid sequence set forth, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment. A method of treating an autoimmune disease or an inflammatory response in a patient by administering the pharmaceutical composition of claim 29 to the patient. A method for detecting B7RP1 in a biological sample, comprising the following steps a) contacting said sample with an antibody of any one of claims 1, 9 and 16 under conditions that the antibody binds to B7RP1; And b) measuring the level of bound antibody in the sample. A nucleic acid molecule encoding the antibody of any one of claims 1, 9 and 16. A host cell comprising the nucleic acid of claim 32. An isolated cell line producing the antibody of any one of claims 1, 9 and 16. A B7RP1 specific binding agent capable of binding to B7RP1, comprising at least one amino acid sequence selected from SEQ ID NOs: 15-40. A pharmaceutical composition comprising a therapeutically effective amount of the binder of claim 35 and a pharmaceutically acceptable carrier. A method of treating an autoimmune disease or an inflammatory response in a patient by administering to the patient the pharmaceutical composition of claim 36. 36. The B7RP1 specific binding agent according to claim 35, which is a protein. A nucleic acid molecule encoding the binder of claim 35. A host cell comprising the nucleic acid of claim 39. An isolated cell line producing the binder of claim 35. A method of treating an autoimmune disease or an inflammatory response in a patient by administering a pharmaceutically effective amount of the binder of claim 35 to the patient. 43. The method of claim 42, wherein the autoimmune disease is rheumatoid arthritis or systemic lupus erythematosus. 43. The method of claim 42, wherein the inflammatory response is asthma. A method for detecting B7RP1 in a biological sample, comprising the following steps a) contacting said sample with the binder of claim 35 under conditions that the binder binds to B7RP1; And b) measuring the level of bound antibody in the sample. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a peptide having the amino acids set forth in any of SEQ ID NOs: 1-39. An isolated antibody that then specifically binds a) the D region of B7RP1, or an antigen binding fragment or immunologically functional immunoglobulin fragment; or b) H region of B7RP1, or antigen binding fragment thereof or immunologically functional immunoglobulin fragment. 48. The antibody of claim 47, wherein the heavy and light chains are joined by a flexible linker to form a single chain antibody. 49. The antibody of claim 48 which is a single chain Fv antibody. 48. The antibody of claim 47 which is a Fab antibody. 48. The antibody of claim 47, wherein the antibody is a Fab 'antibody. 48. The antibody of claim 47, which is a (Fab ') ₂ antibody. 48. The antibody of claim 47, wherein the antibody is a fully human antibody. 48. The antibody of claim 47, wherein the antibody inhibits B7RP1 activity. A method of treating an autoimmune disease or an inflammatory response in a patient by administering to the patient a pharmaceutically effective amount of the antibody of claim 54. 56. The method of claim 55, wherein the autoimmune disease is rheumatoid arthritis or systemic lupus erythematosus. 56. The method of claim 55, wherein the inflammatory response is asthma. A pharmaceutical composition comprising a therapeutically effective amount of the antibody of claim 55 and a pharmaceutically acceptable carrier. 59. A method of treating an autoimmune disease or an inflammatory response in a patient by administering the pharmaceutical composition of claim 58 to the patient. A method for detecting B7RP1 in a biological sample, comprising the following steps a) contacting said sample with the antibody of claim 47 under conditions that the antibody binds to B7RP1; And b) measuring the level of bound antibody in the sample. A nucleic acid molecule encoding the antibody of claim 47. A host cell comprising the nucleic acid of claim 61. An isolated cell line producing the antibody of claim 47. An isolated antibody that is competitive for binding an antibody comprising any of SEQ ID NOs: 1-40. A nucleic acid molecule encoding the antibody of claim 64. A host cell comprising the nucleic acid of claim 65. An isolated cell line producing the antibody of claim 64. An isolated human antibody that specifically binds B7RP1, comprising an amino acid sequence as set forth in any SEQ ID NO: 44-58 or 70-76, or an antigen binding fragment thereof or an immunologically functional immunoglobulin fragment Human antibodies. The antibody of claim 68, wherein the heavy and light chains are joined by a flexible linker to form a single chain antibody. 70. The antibody of claim 69 which is a single chain Fv antibody. The antibody of claim 68, wherein the antibody is a Fab antibody. The antibody of claim 68, wherein the antibody is a Fab 'antibody. The antibody of claim 68, which is a (Fab ') ₂ antibody. The antibody of claim 68, wherein the antibody is a fully human antibody. The antibody of claim 68, wherein the antibody inhibits B7RP1 activity. A method of treating an autoimmune disease or an inflammatory response in a patient by administering a pharmaceutically effective amount of the antibody of claim 75 to the patient. 77. The method of claim 76, wherein the autoimmune disease is rheumatoid arthritis or systemic lupus erythematosus. 77. The method of claim 76, wherein the inflammatory response is asthma. A pharmaceutical composition comprising a therapeutically effective amount of the antibody of claim 75 and a pharmaceutically acceptable carrier. 80. A method of treating a condition caused by increasing the expression of B7RP1 or increasing sensitivity to B7RP1 in a patient by administering the pharmaceutical composition of claim 79. A method for detecting B7RP1 in a biological sample, comprising the following steps a) contacting said sample with the antibody of claim 68 under conditions that the antibody binds to B7RP1; And b) measuring the level of bound antibody in the sample. A nucleic acid molecule encoding the antibody of claim 68. 83. A host cell comprising the nucleic acid of claim 82. An isolated cell line producing the antibody of claim 68. An isolated nucleic acid molecule encoding a polypeptide having an amino acid sequence set forth in any of SEQ ID NOs: 1-40, 44-58 or 70-76. 87. A host cell comprising the nucleic acid of claim 85. 86. An isolated cell line producing the polypeptide of claim 85.

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